WO2024112492A1 - Compositions comprising antibody cleaving enzymes and method of using the same - Google Patents

Abstract

Disclosed herein are compositions for cleaving antibodies and in vivo methods of using these compositions.

Description

COMPOSITIONS COMPRISING ANTIBODY CLEAVING ENZYMES AND METHODS OF USING THE SAME

I. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional App. No. 63/421,996 filed 2 November 2022, U.S. Provisional App. No. 63/492,985 filed 29 March 2023, and U.S. Provisional App. No. 63/502,920 filed 17 May 2023, each of which is incorporated herein in its entirety.

II. STATEMENT REGARDING FEDERAL FUNDING

[0002] This invention was made with Government support under Federal Grant Nos. R01 HL089221, R01 Al 166969, and U19 AI131471 awarded by the National Institute of Health. The Federal Government has certain rights to this invention.

III. REFERENCE TO THE SEQUENCE LISTING

[0003] The Sequence Listing submitted 1 November 2023 as an XML file named “22-2060-WO- Sequence_Listing’‘, created on 31 October 2023 and having a size of 289 kilobytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

IV. BACKGROUND

[0004] Monomeric IgM is the first antibody to emerge as the B cell antigen receptor (BCR) on the surface of B cells followed by secreted pentameric IgM from plasma cells. Class switching upon B cell activation generates different isotypes or subtypes of IgG, which constitute the most abundant, secreted class of antibodies (Engels N, et al. (2018) Immunol Rev. 283: 150-160). Due to the highly diverse BCR repertoire, recognition of self-antigens can sometimes lead to the generation of harmful IgG and IgM autoantibodies leading to autoimmune disease (Suurmond J, et al. (2015) J Clin Invest. 125:2194-2202). Similarly, antibodies directed against human leukocyte antigens (HLAs) expressed on the surface of cells in a transplanted organ can lead to rejection (Choi AY, et al. (2021) Front Immunol. 12:694-763). . Further, while beneficial in the context of immunity against pathogens such as viruses, antibodies against viral vectors such as recombinant adeno-associated viruses (AAV) can preclude administration of gene therapies in prospective patients (Earley J, et al. (2023) Trends Biotechnol. 41 :836-845)). Another pathophysiological manifestation of autoantibodies in the context of autoimmune disease, organ transplantation or gene therapies is the activation of the complement cascade and lytic components thereof, which can lead to significant tissue damage (Goldberg BS, et al. (2020) Immunol Cell Biol. 98:305-317; Grafals M, et al. (2019) Front Immunol. 10:2380; West C, et al. (2023) Hum Gene Ther. 34:554-566; Smith CJ, et al. (2022) Front Immunol. 13:999-1021). Thus, understanding and manipulating IgG and IgM levels can have profound implications in the development of therapeutic modalities with broad clinical impact. [0005] To this end, a wide range of approaches including plasmapheresis, B cell targeting agents, complement inhibitors, neonatal Fc receptor (FcRn) blocking agents and IgG degrading enzymes are being explored to modulate humoral immunity⁷ in autoimmune disease, organ transplant and gene therapy applications (Choi AY, et al. (2021) Front Immunol. 12:694-763; Lee DSW, et al. (2020) Nature Reviews Drug Discov. 20: 179-199; Zelek WM, et al. (2019) Mol Immunol. 114:341-352; Corti M, et al. (2014) Mol Ther Methods Clin Dev. 1 : 14033; Elmore ZC, et al. (2020) JCI Insight. 5(19):el39881; Leborgne C, et al. (2020) Nature Med. 26:1096-1101). Despite this spectrum of agents, strategies that directly address pathogenic IgM and IgG antibodies, which are key players in initiating immune dysfunction, are lacking.

[0006] Thus, there remains an urgent need to generate and characterize antibody-cleaving enzymes, and to develop therapeutics based on those antibody-cleaving enzymes.

V. BRIEF DESCRIPTION OF THE FIGURES

[0007] FIG. 1A - FIG. 1H show structural model and in vitro characterization of a human IgM cleaving enzyme. FIG. 1A shows a representative structural model of the core domain of cysteine protease candidates with active site in orange. FIG. IB confirms the protein expression system and IgM-cleaving assay, we used a known porcine IgM protease from Streptococcus Suis (Idessuis) that is known to cleave porcine IgM, but not human IgM. Immunoblot analyses showing the banding patterns of IgM from human sera following treatment with multiple candidate proteins. A cleavage band was observed for protein candidate protein 2 and 6, hereinafter referred to as IceM and IceM2, respectively. FIG. 1C is a cartoon schematic illustrating predicted cleavage site motif and IgM cleavage product (CP) molecular weights. FIG. ID shows a structural docking analysis of IceM in complex with human IgM heavy⁷ chain (HC) with active site in orange. FIG. IE is an SDS-PAGE gel showing recombinant IceM (41.6 kDa) expression and punfication with BSA loading control. FIG. IF shows an SDS-PAGE gel of purified human IgM, IceM, or IgM treated with 10 pg mL'¹ IceM for 1 hr at 37 °C. FIG. 1G shows a concentration curve for IceM enzymatic activity in human sera. FIG. II and FIG. 1J show an immunoblot analyses showing the banding patterns of IgM, IgA, IgD, IgE, or IgG from human sera (FIG. 1H) and IgM from sera across species (FIG. II) treated with PBS or 10 pg mL"¹ IceM for 1 hr at 37 °C. indicates full length HC (IgM, ~73 kDa; IgG, ~53 kDa; IgA, ~55 kDa IgE, ~73 kDa; IgD, ~63 kDa) and arrows indicate cleavage products. FIG. 1J shows an alignment of IgM sequences from multiple species with the predicted human and NHP cleavage site residues (SEQ ID NO: 110) indicated in the red box. Error bars denote mean ± standard deviation. All experiments were completed in at least biological triplicate. Analyses were performed with GraphPad Prism version 9.5 with P values less than 0.05 being significant. [0008] FIG. 2A - FIG. 2C show structural modeling and analyses of IceM contact residues. FIG. 2A shows structural models showing IdeS with residues in contact with human IgG shown in red. FIG. 2B - FIG. 2C show structural models showing IgM cleaving enzymes IceM (FIG. 2B) and IceM 2 (FIG. 2C) with residues in contact with human IgM shown in red.

[0009] FIG. 3A - FIG. 3C show optimizing linkers for a recombinant IceM fusion protein. FIG. 3A (top) show' structural modeling of IceM fusion proteins with three different linkers (rigid linker, flexible linker 1, and flexible linker 2 with corresponding linker amino acid sequence beneath the structure. Models are colored by pLDDT. FIG. 3A (bottom) show' immunoblot analyses for His-tagged fusion protein expression. The fusion protein with the rigid linker, hereinafter termed IceMG, showed the best expression and was chosen for further evaluation. FIG. 3B show' a structural model of the dual cleaving enzyme IceMG. FIG. 3C shows structural docking analysis of IceMG in complex with human IgM (gray) and human IgG (green) heavy chain with active site in red.

[0010] FIG. 4A - FIG. 4H show in vitro characterization of a human IgM and IgG cleaving enzyme IceMG. FIG. 4A shows aSDS-PAGE gel showing recombinant IceMG (70.4 kDa) expression and purification with BSA loading control. FIG.4B shows a SDS-PAGE gel of purified human IgM. IceMG, or IgM treated with 10 pg mL"¹ IceMG for 1 hr at 37 °C. FIG. 4C shows a schematic illustrating predicted IgG cleavage products (CP). FIG. 4D shows a SDS- PAGE gel of purified human IgG, IceMG, or IgG treated w'ith 10 pg mL"¹ IceMG for 1 hr at 37 °C. FIG. 4E and FIG. 4F shows a concentration curve for IceMG enzymatic activity on IgM (FIG. 4E) or IgG (FIG. 4F) in human sera. FIG. 4G shows immunoblot analyses showing the banding patterns of IgM, IgG, IgA, IgE, or IgD from human sera and IgM or IgG from sera across species treated with PBS or 10 pg mL"¹ IceMG for 1 hr at 37 °C. FIG. 4H show's IgG cleavage site residues for each IgG subtype IgGl (SEQ ID NO: 115), IgG2 (SEQ ID NO: 116), IgG3 (SEQ ID NO: 117), and IgG4 (SEQ ID NO: 118). *indicates full length HC and arrows indicate cleavage products. Error bars denote mean ± standard deviation. All experiments were completed in at least biological triplicate. Analyses were performed with GraphPad Prism version 9.5 with P values less than 0.05 being significant.

[0011] FIG. 5A - FIG. 5G show an in vitro assessment of recombinant IceM and IceMG on B cells. FIG. 5A shows immunoblot analysis of Daudi cells treated with recombinant 10 pg mL"¹ IceM for 1 hr at 37 °C. FIG. 5B show' immunofluorescent staining of surface-bound IgM on Daudi cells. FIG. 5C show representative plots for flow cytometric analysis of IgM surface expression on Daudi cells following treatment with PBS or IceM at 1 hr. Data are gated to analyze the IgM⁺ (FITC⁺) cohort. FIG. 5D show the quantitation of percent IgM⁺ Daudi cells. FIG. 5E are representative plots for flow' cytometric analysis of IgM surface expression on CD 19+ B Cells (BCR) following treatment with PBS, IceM, or IceMG at 1 hr, 12 hr, and 24 hr post-treatment. Data are gated on live cells and then CD19⁺ (APC⁺) to analyze the IgM⁺ (FITC⁺) cohort. FIG. 5F shows the quantitation of percent IgM⁺ B cells from (FIG. 5A). FIG. 5G shows dose curve for IceM enzymatic activity on B cells treated for 1 hr at 37 °C. Error bars denote mean ± standard deviation. All experiments were completed in at least biological triplicate. Statistical significance was determined using an unpaired two-tailed Student’s t-test (FIG. 5D) or two-way ANOVA with Sidak’s correction for multiple comparisons (FIG. 5F). Analyses were performed with GraphPad Prism version 9.5 with P values less than 0.05 being significant.

[0012] FIG. 6A - FIG. 6C shows the in vitro assessment of recombinant IceM and IceMG complement activation. FIG. 6A show- the the illustration of AAV 9- or platelet factor-based complement activation assay. FIG. 6B and FIG. 6C show ELISA based quantitation of C3a levels in human sera treated with PBS or 10 pg mL'¹ IceM for 1 hr at 37 °C to cleave IgM followed by incubation with AAV9 (FIG. 6B) or PF4/Heparin complexes (FIG. 6C). Error bars denote mean ± standard deviation. All experiments were completed in at least biological triplicate. Statistical significance was determined using one-way ANOVA. Analyses were performed with GraphPad Prism version 9.5 with P values less than 0.05 being significant.

[0013] FIG. 7A - FIG. 7N show the in vivo assessment of recombinant IceM and IceMG in passively immunized mice. FIG. 7A show an overview' of experimental design for mouse study. FIG. 7B - FIG. 7E show immunoblots for circulating human IgM in mice treated with PBS of IceM at the dose indicated. FIG. 7F - FIG. 71 show immunoblots for circulating human IgM (top panel) or IgG (bottom panel) in mice treated with PBS of IceM at the dose indicated. Five mice (numbered 1-5) w ere tested per cohort with human serum (Hu) included as a positive control. FIG. 7J show' an overview' of experimental design for AAV mouse study. Luciferase expression was evaluated 21 days post injection in heart and liver tissue for AAV8 (FIG. 7K and FIG. 7L) and AAV9 (FIG. 7M and FIG. 7N). Luciferase expression levels were normalized for total tissue protein concentration and represented as log relative luminescence units per gram of tissue (log RLU/g tissue).

[0014] FIG.8A- FIG.8H show- the Tn vivo assessment of recombinant IceM in rhesus macaques. FIG. 8A show an overview of experimental design for IceM nonhuman primate study. FIG. 8B and FIG. 8C show immunoblot analyses of His-tagged IceM (41.6 kDa) from NHP N303 (FIG. SB) and NHP M741 (FIG. 8C) at various time intervals following IceM administration. FIG. 8D and FIG. 8E show' immunoblots for circulating IgM in animals treated with IceM. Treatment days indicated in red. FIG. 8F shows the quantification of uncleaved IgM and cleavage product bands from immunoblots in FIG. 8G and FIG. 8H. indicates full-length HC and arrows indicate cleavage products. [0015] FIG. 9A - FIG. 9H show the in vivo assessment 1 of recombinant IceMG on circulating IgM in rhesus macaques. FIG. 9A show overview of experimental design for IceMG nonhuman primate study 1. FIG. 9B FIG. 9D (top) show the immunoblot analyses of His-tagged IceMG (70.4 kDa) from animals 49873 (FIG. 9B), 49813 (FIG. 9C), and 499682 (FIG. 9D) at various time intervals following IceMG administration. FIG. 9B - FIG. 9D (bottom) show the immunoblots for circulating IgM in animals treated with IceMG at the dose indicated. FIG. 9E - FIG. 9G show the quantitation of IgM immunoblots from FIG. 9B - FIG. 9D. FIG. 9H shows the ELISA based quantitation of C3a levels in sera from NHPs in FIG. 9B - FIG. 9D following treatment with AAV9 to elicit complement activation, indicates full-length HC and arrows indicate cleavage products.

[0016] FIG. 10A - FIG. 10G shows the in vivo assessment 2 of recombinant IceMG on circulating IgM in rhesus macaques. FIG. 10A show the overview of experimental design for IceMG nonhuman primate study 2 using animals 36725, 37662, 35788, 38131, 38718, 38721. FIG. 10B - FIG. 10D show immunoblot analyses for circulating IgM (FIG. 10B - FIG. 10D) in animals treated with IceMG at the dose indicated. FIG. 10E - FIG. 10G show the quantitation of IgM immunoblots from FIG. 10B - FIG. 10D. * indicates full-length HC and arrows indicate cleavage products.

[0017] FIG. 11A - FIG. 11F show the in vivo assessment of recombinant IceMG on circulating IgG in rhesus macaques. FIG. 11A - FIG. 11F) show immunoblots for circulating IgG in animals treated with IceMG at the dose indicated for nonhuman primate study 1 (FIG. 11A - FIG. HC) or nonhuman primate study 2 (FIG. HD - FIG. HF). *indicates full-length HC and arrows indicate cleavage products.

VI. BRIEF SUMMARY

[0018] Disclosed herein is a recombinant enzyme having IgM specific protease activity. Disclosed herein is a recombinant enz me having IgG specific protease activity⁷. Disclosed herein is a recombinant enzyme having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an enzyme comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46 and having IgM specific protease activity and/or IgG specific protease activity.

[0019] Disclosed herein is a recombinant enzy me that can bind to and cleave at one or more sequences set forth in SEQ ID NO: 111 - SEQ ID NO: 118 in an IgG molecule and at the sequence set forth in SEQ ID NO: 110 in an IgM molecule.

[0020] Disclosed herein is an enzyme comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46. Disclosed herein is an isolated nucleic acid molecule encoding any disclosed recombinant enzy me. Disclosed herein is a codon-optimized isolated nucleic acid molecule encoding any disclosed recombinant enzyme. [0021] Disclosed herein is an isolated nucleic acid molecule comprising the sequence of any one of SEQ ID NO:47 - SEQ ID NO:92. Disclosed herein is an isolated nucleic acid molecule encoding the recombinant enzyme having the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46. Disclosed herein is a vector comprising a nucleic acid sequence encoding the recombinant enzyme comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46. Disclosed herein is a vector comprising a nucleic acid sequence comprising the sequence of any one of SEQ ID NO: 47 - SEQ ID NO: 92 or a fragment thereof.

[0022] Disclosed herein is a method of cleaving immunoglobulin M (IgM) and/or IgM and immunoglobulin G (IgG) the method comprising contacting one or more IgM molecules with an enzyme comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46.

[0023] Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with an enzyme comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46.

[0024] Disclosed herein is a method of reducing the level of circulating intact IgM and/or IgG and IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzy me comprising the sequence of any one of SEQ ID NO: 01 - SEQ ID NO:46.

[0025] Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46. Disclosed herein is a method of reducing and/or minimizing an immune response in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46.

[0026] Disclosed herein is a method reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG specific protease activity, and (ii) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgM specific protease activity.

[0027] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46.

VII. DETAILED DESCRIPTION

[0028] The present disclosure describes formulations, compounded compositions, kits, capsules, containers, and/or methods thereof. It is to be understood that the inventive aspects of which are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0029] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

A. Definitions

[0030] Before the present compounds, compositions, articles, systems, devices, and/or methods are disclosed and descnbed, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0031] This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure.

[0032] As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0033] The phrase “consisting essentially of’ limits the scope of a claim to the recited components in a composition or the recited steps in a method as well as those that do not materially affect the basic and novel characteristic or characteristics of the claimed composition or claimed method. The phrase “consisting of’ excludes any component, step, or element that is not recited in the claim. The phrase “comprising” is synonymous with “including”, “containing”, or “characterized by”, and is inclusive or open-ended. “Comprising” does not exclude additional, unrecited components or steps.

[0034] As used herein, when referring to any numerical value, the term “about” means a value falling within a range that is ± 10% of the stated value.

[0035] Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent '‘about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as ‘'about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0036] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0037] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. In an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.

[0038] As used herein, “isolated” refers to a nucleic acid molecule or a nucleic acid sequence that has been substantially separated, produced apart from, or purified away from other biological components in the cell or tissue of an organism in which the component occurs, such as other cells, chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins. Isolated IceM, IceM2, IceM Full, IceM2 Full, IceM Mid, IceM Lead, IceM2 Lead, Ice MG, IceMG2, IceMG Optilinkerl, IceMG2 Optilinkerl, IceMG-Optimlinker2, and IceMG-Optinlinker3 can be at least 50% pure, such as at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% pure.

[0039] As used herein, the term “subject” refers to the target of administration, e.g., a human being. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g. , cattle, horses, pigs, sheep, goats, etc.), and laboratory' animals (e.g. , mouse, rabbit, rat, guinea pig, fruit fly, etc.). Thus, the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex, and thus, adult and child subjects, as well as fetuses, whether male or female, are intended to be covered. In an aspect, a subject can be a human patient. In an aspect, a subject can have a disease or disorder, be suspected of having a disease or disorder, or be at risk of developing a disease or disorder (e.g., a genetic disease or disorder). In an aspect, a subject can have an AD or AIF disease and/or disorder. In an aspect, a subject can be in need of one or more transplanted organs. In an aspect, a subject can have received one or more transplanted organs. In an aspect, a subject can have received one or more transplanted organs. In an aspect, a subject can be in need of gene therapy, can be receiving gene therapy, or can have received gene therapy.

[0040] As used herein, the term “diagnosed” means having been subj ected to an examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the disclosed enzymes, the disclosed recombinant enzymes, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed pharmaceutical formulations, or any combination thereof, or by one or more of the disclosed methods. For example, “diagnosed with a disease or disorder” means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as a genetic disease or disorder) that can be treated by one or more of the disclosed enzymes, the disclosed recombinant enzymes, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed pharmaceutical formulations, or any combination thereof, or by one or more of the disclosed methods. For example, “suspected of having a disease or disorder” can mean having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition (such as a genetic disease or disorder) that can likely be treated by one or more of the disclosed enzymes, the disclosed recombinant enzymes, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed pharmaceutical formulations, or any combination thereof, or by one or more of the disclosed methods. In an aspect, an examination can be physical, can involve various tests (e.g., blood tests, genotyping, biopsies, etc.) and assays (e.g., enzymatic assay), or a combination thereof.

[0041] A “patient” refers to a subject afflicted with a disease or disorder (e.g., a genetic disease or disorder). In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder. In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder and is seeking treatment or receiving treatment for a disease or disorder. In an aspect, a patient can have an AD or AIF disease and/or disorder. In an aspect, a patient can be in need of one or more transplanted organs. In an aspect, a patient can have received one or more transplanted organs. In an aspect, a patient can have received one or more transplanted organs. In an aspect, a patient can be in need of gene therapy, can be receiving gene therapy, or can have received gene therapy.

[0042] As used herein, the phrase "‘identified to be in need of treatment for a disease or disorder,” or the like, refers to selection of a subject based upon need for treatment of the disease or disorder. For example, a subject can be identified as having a need for treatment of a disease or disorder (e.g., a genetic disease or disorder, an AD, an AIF disease, transplanted organs) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the genetic disease or disorder. In an aspect, the identification can be performed by a person different from the person making the diagnosis. In an aspect, the administration can be performed by one who performed the diagnosis.

[0043] As discussed herein, humoral immunity is mediated by circulating proteins called antibodies, which are produced by B lymphocytes. Antibodies exist either as membrane-bound antibodies on the surface of B lymphocytes that function as receptors for the antigen or as secreted antibodies that reside in the circulation, tissues, and mucosal sites to neutralize toxins, prevent the entry and spread of pathogens, and eliminate microbes. The activation of B lymphocytes is initiated by the specific recognition of antigens by the surface Ig receptors of these cells. Activation leads to proliferation of antigen-specific cells and their differentiation in cooperation with Th cells, generating memory B lymphocytes and antibody-secreting plasma cells. The type and amount of antibodies produced vary according to the antigen driving the immune response, the involvement of T lymphocytes, a prior history of antigen exposure, and the site where activation occurs. Antibody responses to protein antigens require that the specifically recognized antigen is internalized by B lymphocytes and that a peptide fragment of it is presented to CD4+ helper T lymphocytes that subsequently activate these B lymphocytes. Because of the involvement of helper T lymphocytes, this type of response is called ‘T-dependent antibody response' (TDAR). In contrast, antibody responses to multivalent nonproteinic antigens with repeating determinants, such as polysaccharides, some lipids, and nucleic acids, do not involve antigen-specific helper T lymphocytes and are called ‘T-independent antibody responses.’ In TDARs, plasma cells or their precursors migrate from germinal centers in the peripheral lymphoid organs, where they are produced, to the bone marrow, where they live for many years and ensure specific protection against microbes for long periods of time.

[0044] As used herein, ‘’immune response” refers to the body’s response to foreign agents and infections organisms and includes (i) the body’s innate or non-specific immune system and (ii) the body's adaptive or specific immune system (including B cells, cytotoxic T cells, and helper T cells). [0045] As used herein, ‘'primary and secondary antibody responses” to protein antigens differ qualitatively and quantitatively. Primary responses result from the activation of naive B lymphocytes that have not been previously stimulated by the antigen, whereas a secondary' immune response is elicited when the same antigen stimulates antigen-specific memory B lymphocytes. In secondary responses as compared to primary responses, antibody production is greater and peak antibody levels attained more rapidly (approximately 2-3 vs. 7 days), the main isotype is IgG instead of IgM, and antibody affinity is much greater. Once released, the various forms of antibodies (Ig) possess a number of effector functions to engage the antigen. Microbes, tumor cells, or foreign proteins can express several types of antigens and multiple copies of these antigens. If the microbial antigen to which an antibody is directed is associated with the toxic portion of a molecule, then the antibody can neutralize the toxin. The production of neutralizing antibodies is frequently a problem in the therapeutic application of biotechnolog -derived recombinant proteins as neutralizing antibodies can become a rate-limiting step during preclinical testing because animals may perceive the recombinant protein, especially when humanized, as non-self.

[0046] Most of the effector functions of humoral immunity are mediated by antibody-activated processes, but they are also key players in innate immunity. For example, the classical activation pathway of the complement cascade is triggered by antigen-antibody complexes and is specifically mediated by the constant region of the Ig molecule. Both IgM and IgG can activate the complement system, which results in the biologically active components identified previously, including the lytic unit, the chemotactic factors, and the complement peptides, which opsonize the microbe to facilitate phagocytosis. IgM and IgG can function to opsonize some microbes independent of complement activation in an antigen-dependent fashion because macrophages and neutrophils have receptors on their surface, which recognize the constant region of Ig (Fc receptors, FcRs). FcRs also play a maj or role in the ability' of IgG to participate in a process known as ‘antibody-dependent cellular cytotoxicity,’ whereby antigen-specific antibody attaches via FcRs to certain types of cells, including NK. cells, enabling these cells to attach intimately to the target cell and trigger cell death. Finally, FcRs are also the primary effector mechanism for IgE, which is the principal immune defense against certain types of parasitic infections (most notably, helminths) and is produced primarily by the external immune system along secretory surfaces. IgE binds to FcR on the surface of mast cells and basophils. Once bound to FcR, IgE can sen e as an antigen-specific receptor to trigger the release of proinflammatory factors, including vasoactive amines (e.g., histamine) and products of the arachidonic cascade (e.g., leucotrienes and prostaglandins). Systemic immunity is mediated by IgM and IgG, the latter being the major form of Ig found in the blood. Local immunity is mediated primarily by IgA and IgE. [0047] As known to the skilled person in the art, autoimmune disease (AD) originates in the adaptive immune system. AD occurs when adaptive immune cells lose their ability to maintain self-tolerance of human cells. As a result, the immune system begins attacking healthy tissues as though they were infectious agents. Some ADs are organ-specific, whereas others can affect multiple parts of the body. Recurrent fever and chronic inflammation can be attributed to autoimmune disease. However, many possible symptoms are unique to AD, including hair loss, dry mouth, temperature sensitivity’, muscle weakness, reproductive issues, and more. A variety of conditions including environment, hormones, injury', infection, and genes can all play a role in initiating autoimmunity. AD is thought to be triggered by a combination of multiple factors not limited to genetics. Although the exact causes remain unknown, researchers believe that a variety of conditions including environment, hormones, injury, infection, and genes could all play a role in initiating autoimmunity. Autoimmune conditions are quite common. Current data estimates that ADs affect at least 14 million people in the U.S. alone. There are 80-100 known autoimmune and autoimmune-related conditions, including Hashimoto’s thyroiditis. Type 1 diabetes, Sjogren’s, and celiac disease. Gender prevalence in AD is well documented. In fact, around 80% of those diagnosed are women. While ADs can affect anyone at any age, the majority' tend to appear during adulthood.

[0048] Autoinflammatory (AIF) diseases only involve the innate immune system. AIFs occur when innate immune cells are activated without an infection or injury being present. This mishap kickstarts the release of cytokines and other immune responses, causing fever and inflammation. Recurring episodes of high fever are the primary' symptom of AIF. Similar to some autoimmune diseases, autoinflammatory symptoms can be widespread, affecting the joints, gastrointestinal tract, skin, eyes, and internal organs.

[0049] As used herein, a "regulatory element” can refer to promoters, enhancers, internal ribosomal entry' sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Regulatory elements can include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory' sequences).

[0050] As used herein, "inhibit." “inhibiting”, and “inhibition” mean to diminish or decrease an activity, level, response, condition, severity, disease, or other biological parameter (such as the level of circulating IgG and/or circulating IgM or the level of surface-bound IgM on B cells). This can include, but is not limited to, the complete ablation of the activity', level, response, condition, severity', disease, or other biological parameter (such as the level of circulating IgG and/or IgM or the level of surface-bound IgM on B cells). This can also include, for example, a 10% inhibition or reduction in the activity, level, response, condition, severity, disease, or other biological parameter as compared to the native or control level (e.g., a subj ect not having a disease or disorder such as a genetic disease or disorder). Thus, in an aspect, the inhibition or reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of reduction in between as compared to native or control levels. In an aspect, the inhibition or reduction can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% as compared to native or control levels. In an aspect, the inhibition or reduction can be 0-25%, 25-50%, 50-75%, or 75- 100% as compared to native or control levels. In an aspect, a native or control level can be a predisease or pre-disorder level.

[0051] The words "treat" or “treating’’ or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In an aspect, the terms cover any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease. For example, in an aspect, treating a disease or disorder can reduce the severity of an established a disease or disorder in a subject by l%-100% as compared to a control (such as, for example, an individual not having a genetic disease or disorder). In an aspect, treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a disease or disorder (such as a genetic disease or disorder). For example, treating a disease or disorder can reduce one or more symptoms of a disease or disorder in a subject by 1 %-l 00% as compared to a control (such as, for example, an individual not having a genetic disease or disorder). In an aspect, treating can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms of an established a disease or disorder. It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of a disease or disorder. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of a disease or disorder.

[0052] As used herein, the term “prevent” or “preventing” or “prevention” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing a disease or disorder having chromatin deregulation and/or chromatin dysregulation is intended. The words “prevent”, “preventing”, and “prevention” also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., an individual) not having a given a disease or disorder (such as a genetic disease or disorder) or related complication from progressing to that complication.

[0053] As used herein, the terms “administering” and “administration” refer to any method of providing one or more of the disclosed enzymes, the disclosed recombinant enzymes, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed pharmaceutical formulations, or any combination thereof to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, the following: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV). Administration of a disclosed enzy me, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical composition, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed small molecule, a disclosed endonuclease, a disclosed oligonucleotide, and/or a disclosed RNA therapeutic can comprise administration directly into the CNS or the PNS. Administration can be continuous or intermittent. Administration can comprise a combination of one or more route.

[0054] In an aspect, the skilled person can determine an efficacious dose, an efficacious schedule, and an efficacious route of administration for the disclosed enzymes, the disclosed recombinant enzymes, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed pharmaceutical formulations, or any combination thereof to treat or prevent a disease or disorder (such as genetic disease or disorder). In an aspect, the skilled person can also alter, change, or modify an aspect of an administering step to improve efficacy of the disclosed enzymes, the disclosed recombinant enzy mes, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed pharmaceutical formulations, or any combination thereof. [0055] By '‘determining the amount” is meant both an absolute quantification of a particular analyte (e.g., level of IgG and/or IgM) or a determination of the relative abundance of a particular analyte (e.g., level of IgG and/or IgM compared to a control / reference level). The phrase includes both direct or indirect measurements of abundance or both.

[0056] As used herein. IdeZ is a cysteine protease that inactivates IgG antibodies by cleaving IgG at the lower hinge region of the heavy chain producing one F(ab’)? and one homodimeric Fc fragment.

[0057] As used herein, the term “pharmaceutically acceptable carrier'’ refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. In an aspect, a pharmaceutical carrier employed can be a solid, liquid, or gas. In an aspect, examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. In an aspect, examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water. In an aspect, examples of gaseous carriers can include carbon dioxide and nitrogen. In preparing a disclosed composition for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcry stalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polygly colide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial -retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0058] As used herein, the term '‘excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See, also, for reference. Remington’s Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, Pa., which is hereby incorporated by reference in its entirety.

[0059] As used herein, “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.

[0060] The term “contacting” as used herein refers to bringing one or more of the disclosed enzymes, the disclosed recombinant enzymes, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed pharmaceutical formulations, or any combination thereof together with a target area or intended target area (e.g. a cleavage site on IgG (SEQ ID NO: 111 - SEQ ID NO: 118) and/or IgM (SEQ ID NO: 110)) in such a manner that the one or more of the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations, or a combination thereof exert an effect on the intended target or targeted area either directly or indirectly. A target area can comprise one or more cells, and in an aspect, one or more cells can be in a subject. A target area or intended target area can be one or more of a subject’s organs (e.g., lungs, heart, liver, kidney, brain, etc.). In an aspect, a target area or intended target area can be any organ, tissue, or cells that are affected by a disease or disorder (such as a genetic disease or disorder).

[0061] As used herein, “determining” can refer to measuring or ascertaining the presence and severity of a disease or disorder, such as, for example, a genetic disease or disorder. Methods and techniques used to determine the presence and/or severity of a disease or disorder are ty pically known to the medical arts. For example, the art is familiar with the ways to identify and/or diagnose the presence, severity, or both of a disease or disorder (such as, for example, a genetic disease or disorder).

[0062] As used herein, “effective amount'’ and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and/or prevention of a disease or disorder (e.g., a genetic disease or disorder) or a suspected disease or disorder. As used herein, the terms “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired an effect on an undesired condition (e.g., a disease or disorder). For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. In an aspect, “therapeutically effective amount” means an amount of a disclosed enzy me, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation; that (i) treats the particular disease, condition, or disorder (e.g., a genetic disease or disorder), (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder e.g., a genetic disease or disorder), or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein (e.g., a genetic disease or disorder). The specific therapeutically effective dose level for any particular patient will depend upon a variety' of factors including the disorder being treated and the severity of the disorder; the disclosed enzymes, the disclosed recombinant enzymes, the disclosed isolated nucleic acid molecules, disclosed vectors, disclosed pharmaceutical formulations employed; the disclosed methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations employed; the duration of the treatment; drugs used in combination or coincidental with the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations employed, and other like factors well known in the medical arts. For example, it is within the skill of the art to start doses of the disclosed enzymes, the disclosed recombinant enzymes, the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, then the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, a single dose of the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition, such as, for example, a disease or disorder due to a missing, deficient, and/or mutant protein or enzyme.

[0063] As used herein. “RNA therapeutics” can refer to the use of oligonucleotides to target RNA. RNA therapeutics can offer the promise of uniquely targeting the precise nucleic acids involved in a particular disease with greater specificity, improved potency, and decreased toxicity. This could be particularly powerful for genetic diseases where it is most advantageous to aim for the RNA as opposed to the protein. In an aspect, a therapeutic RNA can comprise one or more expression sequences. As known to the art, expression sequences can comprise an RNAi, shRNA, mRNA, non-coding RNA (ncRNA), an antisense such as an antisense RNA, miRNA, morpholino oligonucleotide, peptide-nucleic acid (PNA) or ssDNA (with natural, and modified nucleotides, including but not limited to, LNA, BNA, 2’-O-Me-RNA, 2 -MEO-RNA, 2'-F-RNA), or analog or conjugate thereof. In an aspect, a disclosed therapeutic RNA can comprise one or more long non-coding RNA (IncRNA), such as, for example, along intergenic non-coding RNA (lincRNA), pre-transcript, pre-miRNA, pre-mRNA, competing endogenous RNA (ceRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), pseudo-gene, rRNA, or tRNA. In an aspect, ncRNA can be piwi-interacting RNA (piRNA), primary miRNA (pri-miRNA), or premature miRNA (pre-miRNA). In an aspect, a disclosed therapeutic RNA or an RNA therapeutic can comprise antisense oligonucleotides (ASOs) that inhibit mRNA translation, oligonucleotides that function via RNA interference (RNAi) pathway, RNA molecules that behave like enzymes (ribozymes), RNA oligonucleotides that bind to proteins and other cellular molecules, and ASOs that bind to mRNA and form a structure that is recognized by RNase H resulting in cleavage of the mRNA target. In an aspect, RNA therapeutics can comprise RNAi and ASOs that inhibit mRNA translation. Generally speaking, as know n to the art, RNAi operates sequence specifically and post-transcriptionally by activating ribonucleases which, along with other enzymes and complexes, coordinately degrade the RNA after the original RNA target has been cut into smaller pieces while antisense oligonucleotides bind to their target nucleic acid via Watson-Crick base pairing, and inhibit or alter gene expression via steric hindrance, splicing alterations, initiation of target degradation, or other events.

[0064] As used herein, “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein must contain at least two amino acids and there is no limitation on the maximum number of amino acids that can comprise a protein’s sequence. The term “peptide” can refer to a short chain of amino acids including, for example, natural peptides, recombinant peptides, synthetic peptides, or any combination thereof. Proteins and peptides can include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others.

[0065] "Nucleic acid” or "oligonucleotide” or "polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand can also define the sequence of the complementary strand. Thus, a nucleic acid can encompass the complementary strand of a depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid can encompass substantially identical nucleic acids and complements thereof. A single strand can provide a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid can encompass a probe that hybridizes under stringent hybridization conditions. A nucleic acid can be single-stranded, or double-stranded, or can contain portions of both double-stranded and single-stranded sequence. The nucleic acid can be DNA, both genomic and cDN A, RN A, or a hybrid, where the nucleic acid can contain combinations of deoxy ribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods. Also as used herein, the terms “nucleic acid,” “nucleic acid molecule,” "nucleic acid construct,” "nucleotide sequence”, and "polynucleotide” can refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term can encompass RNA/DNA hybrids. When dsRNA is produced synthetically, less common bases, such as inosine, 5- methylcytosine, 6-methyladenine. hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2'-hy droxy in the ribose sugar group of the RNA can also be made. A "synthetic” nucleic acid or polynucleotide, as used herein, refers to a nucleic acid or polynucleotide that is not found in nature but is constructed by the hand of man and therefore is not a product of nature.

[0066] A “polynucleotide” is a sequence of nucleotide bases, and may be RNA, DNA, or DNA- RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotides).

[0067] A "fragment” or "portion” of a nucleotide sequence can be understood to mean a nucleotide sequence of reduced length relative (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment or portion according to the disclosure can be. where appropriate, included in a larger polynucleotide of which it is a constituent. In an aspect, a fragment or portion of a nucleotide sequence or nucleic acid sequence can comprise the sequence encoding an exon having one or more mutations.

[0068] A “fragment’' or “portion” of an amino acid sequence can be understood to mean an amino acid sequence of reduced length relative (e.g., reduced by 1, 2. 3, 4, 5. 6, 7, 8. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more ammo acids) to a reference amino acid sequence and comprising, consisting essentially of, or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference amino acid sequence. Such an amino acid fragment or portion according to the disclosure can be, where appropriate, included in a larger amino acid sequence of which it is a constituent.

[0069] “Complement” or “complementary” as used herein means a nucleic acid can mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. “Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

[0070] As used herein, “operably linked” means that expression of a gene or a transgene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5’ (upstream) or 3’ (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0071 ] As used herein, “promoter” or “promoters” are known to the art. Depending on the level and tissue-specific expression desired, a variety of promoter elements can be used. A promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native (endogenous) or foreign (exogenous) and can be a natural or a synthetic sequence. By foreign or exogenous, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. [0072] '‘Tissue-specific promoters” are known to the art and include, but are not limited to, neuron-specific promoters, muscle-specific promoters, liver-specific promoters, skeletal musclespecific promoters, and heart-specific promoters.

[0073] “Liver-specific promoters” are known to the art and include, but are not limited to, the thyroxin binding globulin (TBG) promoter, the al-microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter, the human albumin (hALB) promoter, the thyroid hormone- binding globulin promoter, the a- 1 -anti -trypsin promoter, the bovine albumin (bAlb) promoter, the murine albumin (mAlb) promoter, the human al -antitrypsin (hAAT) promoter, the ApoEhAAT promoter comprising the ApoE enhancer and the hAAT promoter, the transthyretin (TTR) promoter, the liver fatty acid binding protein promoter, the hepatitis B virus (HBV) promoter, the DC172 promoter comprising the hAAT promoter and the al -microglobulin enhancer, the DC 190 promoter comprising the human albumin promoter and the prothrombin enhancer, or any other natural or synthetic liver-specific promoter. In an aspect, a liver specific promoter can comprise about 845-bp and comprise the thyroid hormone-binding globulin promoter sequences (2382 to 13), two copies of al-microglobulin/bikunin enhancer sequences (22,804 through 22,704), and a 71-bp leader sequence as described by Ill CR, et al. (1997).

[0074] Ubiquitous/constitutive promoters” are known to the art and include, but are not limited to, a CMV major immediate-early enhancer/chicken beta-actin promoter, a cytomegalovirus (CMV) major immediate-early promoter, an Elongation Factor 1-a (EFl-a) promoter, a simian vacuolating virus 40 (SV40) promoter, an AmpR promoter, a PyK promoter, a human ubiquitin C gene (Ubc) promoter, a MFG promoter, a human beta actin promoter, a CAG promoter, a EGR1 promoter, a FerH promoter, a FerL promoter, a GRP78 promoter, a GRP94 promoter, a HSP70 promoter, a 0-kin promoter, a murine phosphoglycerate kinase (mPGK.) or human PGK (hPGK) promoter, a ROSA promoter, human Ubiquitin B promoter, a Rous sarcoma virus promoter, or any other natural or synthetic ubiquitous/constitutive promoters.

[0075] As used herein, an “inducible promoter” refers to a promoter that can be regulated bypositive or negative control. Factors that can regulate an inducible promoter include, but are not limited to, chemical agents (e g., the metallothionein promoter or a hormone inducible promoter), temperature, and light.

[0076] As used herein, the term “seroty pe” is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness can be determined by the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes). [0077] As used herein, “tropism’’ refers to the specificity of an AAV capsid protein present in an AAV viral particle, for infecting a particular type of cell or tissue. The tropism of an AAV capsid for a particular type of cell or tissue may be determined by measuring the ability of AAV vector particles comprising the hybrid AAV capsid protein to infect or to transduce a particular type of cell or tissue, using standard assays that are well-known in the art such as those disclosed in the examples of the present application. As used herein, the term “liver tropism” or “hepatic tropism” refers to the tropism for liver or hepatic tissue and cells, including hepatocytes.

[0078] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they are optimally aligned. For example, sequence similarity or identity can be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences can have “substantial sequence identity” if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, preferably 90%, 95%, 98%, 99% or more. Such sequences are also referred to as “variants” herein, e.g., other variants of a missing, deficient, and/or mutant protein or enzyme. It should be understood that sequence with substantial sequence identity do not necessarily have the same length and may differ in length. For example, sequences that have the same nucleotide sequence but of which one has additional nucleotides on the 3’- and/or 5’-side are 100% identical.

[0079] A “heterologous” or a “recombinant” nucleotide or amino acid sequence as used interchangeably herein can refer to a nucleotide or an amino acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide or amino acid sequence.

[0080] Different nucleic acids or proteins having homology' can be referred to as “homologues”. The term homologue includes homologous sequences from the same and other species and orthologous sequences from the same and other species. “Homology” refers to the level of similarity' betw een two or more nucleic acid and/or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology' also refers to the concept of similar functional properties among different nucleic acids or proteins. Thus, the disclosed compositions and disclosed methods can comprise homologues to the disclosed nucleotide sequences and/or disclosed polypeptide sequences.

[0081] “Orthologous,” as used herein, can refer to homologous nucleotide sequences and/or amino acid sequences in different species that arose from a common ancestral gene during speciation. A homologue of a disclosed nucleotide sequence or a disclosed polypeptide can have substantial sequence identity (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%) to a disclosed nucleotide sequence or a disclosed polypeptide.

[0082] As used herein, “codon optimization” can refer to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing one or more codons or more of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. As contemplated herein, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database.” Many methods and software tools for codon optimization have been reported previously . (See, for example, genomes .urv.es/ OPTIMIZER/) .

[0083] For example, a codon is a sequence of three nucleotides that encodes for a specific amino acid in the synthesis of a protein. There are 64 distinct codons, but only 20 amino acids, leading to the degeneracy of the genetic code. For instance, the amino acid Leucine can be encoded with six synonymous codons CUU, CUC, CUA, CUG, UUA and UUG whereas Cysteine can be encoded with two codons UGU and UGC. Overall, 2 of the 20 amino acids can be encoded with 1 codon, 9 with 2 codons, 1 with 3 codons, 5 with 4 codons and 3 with 6 codons leading to 61 essential codons. The remaining three codons are stop codons and are reserved for termination of protein formation. Codon degeneracy leads to many possible ways of encoding a protein, e.g. a typical 375-amino acid protein in humans can be potentially encoded by 10207 different codon sequences. All possible encodings and resulting sequences are not equally likely to be observed in nature; however, as some synonymous codons are more frequently used than others in encoding a particular amino acid in a particular organism. This phenomenon is called ‘codon usage bias’ or ‘codon bias’. As an example, Leucine is encoded 39.5% of the time with codon CUG in Homo sapiens, whereas the same codon is used 11.1% of the same amino acid’s encoding in Saccharomyces cerevisiae (Nakamura et al., 2000).

[0084] As used herein, “immune tolerance,” “immunological tolerance,” and “immunotolerance” refers to a state of unresponsiveness or blunted response of the immune system to substances (e.g., protein replacement therapy, enzyme replacement therapy, recombinant product, a transplanted organ, a vector protein, etc.) that have the capacity to elicit an immune response in a subject. Immune tolerance is induced by prior exposure to a specific antigen. Immune tolerance can be determined in a subject by measuring antibodies against a particular antigen (such as, for example, to an HLA on a transplanted organ, an enzy me or therapeutic protein as part of replacement therapy, a viral vector, an AAV capsid protein). Low or absent antibody titers over time is an indicator of immune tolerance or the efficacy of a disclosed enz me or disclosed recombinant enzy me. For example, immune tolerance can be established by having IgG antibody titers of less than or equal to about 12,000, 11,500, 11,000. 10.500, 10,000, 9,500. 9,000, 8,500, 8,000, 7.500, 7,000, 6.500, or 6,000 following gene therapy (such as the administration of the transgene encoding, for example, a missing, deficient, and/or mutant protein or enzyme), protein replacement therapy, enzyme replacement therapy, or a recombinant product.

[0085] As known to the art, antibodies (Abs) can mitigate AAV infection through multiple mechanisms by binding to AAV capsids and blocking critical steps in transduction such as cell surface attachment and uptake, endosomal escape, productive trafficking to the nucleus, or uncoating as well as promoting AAV opsonization by phagocytic cells, thereby mediating their rapid clearance from the circulation. For example, in humans, serological studies reveal a high prevalence of NAbs in the worldwide population, with about 67% of people having antibodies against AAV1, 72% against AAV2, and approximately 40% against AAV serotypes 5 through 9. Vector immunogenicity represents a major challenge in re-administration of AAV vectors.

[0086] In an aspect, also disclosed herein are partial self-complementary⁷ parvovirus (e.g., a disclosed AAV) genomes, plasmid vectors encoding the parvovirus genomes, and parvovirus (e.g., a disclosed AAV) particles including such genomes. In an aspect, provided herein is a plasmid vector comprising a nucleotide sequence encoding a disclosed parvovirus genome such as for example, a disclosed AAV. In an aspect, provided herein is a partial self-complementary' parvovirus genome including a payload construct, parvovirus ITRs flanking the payload construct, and a self-complementary region flanking one of the ITRs. A self-complementary⁷ region can comprise a nucleotide sequence that is complementary⁷ to the payload construct. A disclosed self- complementary region can have a length that is less the entire length of the pay load construct.

[0087] In an aspect, a disclosed self-complementary⁷ region of a disclosed parvovirus genome can comprise a minimum length, while still having a length that is less the entire length of the payload construct. In an aspect, a disclosed self-complementary region can comprise at least 50 bases in length, at least 100 bases in length, at least 200 in length, at least 300 bases in length, at least 400 bases in length, at least 500 bases in length, at least 600 bases in length, at least 700 bases in length, at least 800 bases in length, at least 900 bases in length, or at least 1,000 bases in length.

[0088] In an aspect, a “self-complementary parvovirus genome” can be a single stranded polynucleotide having, in the 5’ to 3’ direction, a first parvovirus ITR sequence, a heterologous sequence (e.g., payload construct comprising, for example, a desired gene), a second parvovirus ITR sequence, a second heterologous sequence, wherein the second heterologous sequence is complementary to the first heterologous sequence, and a third parvovirus ITR sequence. In contrast to a self-complementary genome, a "partial self-complementary genome” does not include three parvovirus ITRs and the second heterologous sequence that is complementary to the first heterologous sequence has a length that is less than the entire length of the first heterologous sequence (e.g., payload construct). Accordingly, a partial self-complementary genome is a single stranded polynucleotide having, in the 5’ to 3’ direction or the 3’ to 5’ direction, a first parvovirus ITR sequence, a heterologous sequence (e.g., payload construct), a second parvovirus ITR sequence, and a self-complementary region that is complementary to a portion of the heterologous sequence and has a length that is less than the entire length the heterologous sequence.

[0089] As used herein, “immune-modulating” refers to the ability of a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a disclosed agent to alter (modulate) one or more aspects of the immune system. The immune system functions to protect the organism from infection and from foreign antigens by cellular and humoral mechanisms involving lymphocytes, macrophages, and other antigen-presenting cells that regulate each other by means of multiple cell-cell interactions and by elaborating soluble factors, including lymphokines and antibodies, that have autocrine, paracrine, and endocrine effects on immune cells.

[0090] As used herein, “immune modulator” refers to an agent that is capable of adjusting a given immune response to a desired level (e.g., as in immunopotentiation, immunosuppression, or induction of immunologic tolerance). Examples of immune modulators include but are not limited to, a disclosed immune modulator can comprise aspirin, azathioprine, belimumab, betamethasone dipropionate, betamethasone valerate, bortezomib, bredinin, cyazathioprine, cyclophosphamide, cyclosporine, deoxyspergualin, didemnin B, fluocinolone acetonide, folinic acid, ibuprofen. IL6 inhibitors (such as sarilumab) indomethacin, inebilizumab, intravenous gamma globulin (IVIG), methotrexate, methylprednisolone, mycophenolate mofetil, naproxen, prednisolone, prednisone, prednisolone indomethacin, rapamycin, rituximab, sirolimus, sulindac, synthetic vaccine particles containing rapamycin (SVP-Rapamycin or ImmTOR), thalidomide, tocilizumab, tolmetin, triamcinolone acetonide. anti-CD3 antibodies, anti-CD4 antibodies, anti-CD19 antibodies, anti- CD20 antibodies, anti-CD22 antibodies, anti-CD40 antibodies, anti-FcRN antibodies, anti-IL6 antibodies, anti-IGFIR antibodies, an IL2 mutein, a BTK inhibitor, or a combination thereof. In an aspect, a disclosed immune modulator can comprise one or more Treg (regulatory T cells) infusions (e.g., antigen specific Treg cells to AAV). In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, an immune modulator can be administered by any suitable route of administration including, but not limited to, in utero, intra-CSF, intrathecally, intravenously, subcutaneously, trans dermally, intradermally, intramuscularly, orally, trans cutaneously, intraperitoneally (IP), or intravaginally. In an aspect, a disclosed immune modulator can be administered using a combination of routes. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV). Administration of an immune modulator can be continuous or intermittent, and administration can comprise a combination of one or more routes.

[0091] As used herein, the term "immunotoleranf ’ refers to unresponsiveness to an antigen (e.g., a vector, a therapeutic protein, a transgene product, etc.). An immunotolerant promoter can reduce, ameliorate, or prevent transgene-induced immune responses that can be associated with gene therapy. Assays known in the art to measure immune responses, such as immunohistochemical detection of cytotoxic T cell responses, can be used to determine whether one or more promoters can confer immunotolerant properties.

[0092] As used herein, the term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

[0093] As used herein, the term “in combination” in the context of the administration of other therapies (e.g., other agents) includes the use of more than one therapy (e.g., drug therapy). Administration “in combination with” one or more further therapeutic agents includes simultaneous (e.g.. concurrent) and consecutive administration in any order. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. By way of non-limiting example, a first therapy (e.g., a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof) may be administered prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks), concurrently, or after (e.g., 1 minute. 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours. 12 hours, 24 hours, 48 hours, 72 hours, 96 hours. 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, or 12 weeks or longer) the administration of a second therapy (e.g., gene therapy, enzyme replacement therapy, protein replacement therapy, recombinant product) to a subject having or diagnosed with a disease or disorder (such as a genetic disease or disorder).

[0094] Disclosed are the components to be used to prepare the disclosed enzymes, the disclosed recombinant enzymes, disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations as well as the disclosed enzymes, the disclosed recombinant enzymes, the disclosed isolated nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B. and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B- F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

B. Compositions for Transcriptome Engineering

1. Enzymes

[0095] Disclosed herein is an enzy me having IgM specific protease activity. Disclosed herein is an enzyme having IgG specific protease activity. Disclosed herein is an enzyme having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme having IgM specific protease activity. Disclosed herein is a recombinant enzyme having IgG specific protease activity. Disclosed herein is a recombinant enzyme having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a fusion protein having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme that can bind to and cleave at one or more sequences set forth in SEQ ID NO: 111 - SEQ ID NO: 118 and at the sequence set forth in SEQ ID NO: 110.

[0096] Disclosed herein is an enzy me comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46. Disclosed herein is an enzyme comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46 and having IgM specific protease activity and/or IgG specific protease activity.

[0097] Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO:01. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO:04. [0098] Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO:07. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 10. Disclosed herein is an enzy me comprising the sequence set forth in SEQ ID NO: 13.

[0099] Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 16. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 19.

[0100] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO: 02 or SEQ IDNO:03 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03 or a fragment thereof and having IgM specific protease activity.

Disclosed herein is a recombinant enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03 or a fragment thereof and having IgM specific protease activity.

[0101] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity’ to the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity' to the sequence set forth in SEQ ID NO: 05 or SEQ IDNO:06 and having IgM specific protease activity'. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06 or a fragment thereof and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06 or a fragment thereof and having IgM specific protease activity.

[0102] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity⁷, or at least 90% identity⁷ to the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme compnsing a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity⁷ to the sequence set forth in SEQ ID NO: 08 or SEQ ID NO:09 and having IgM specific protease activity'. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09 or a fragment thereof and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity⁷ to the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09 or a fragment thereof and having IgM specific protease activity.

[0103] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity' to the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity' to the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12 and having IgM specific protease activity. Disclosed herein is a recombinant enzy me comprising the sequence set forth in SEQ ID NO:11 or SEQ ID NO: 12 or a fragment thereof and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12 or a fragment thereof and having IgM specific protease activity.

[0104] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity’ to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%. at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15 and having IgM specific protease activity. Disclosed herein is a recombinant enzy me comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15 or a fragment thereof and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%. at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15 or a fragment thereof and having IgM specific protease activity.

[0105] Disclosed herein is a recombinant fusion protein comprising a proteolytic domain from a disclosed IgG specific protease linked to a proteolytic domain from a disclosed IgM specific protease. In an aspect of a disclosed recombinant fusion protein, a linker can comprise a flexible linker or a rigid linker. In an aspect, a disclosed linker can comprise the sequence of any one of SEQ ID NO:94 - SEQ ID NO: 104. In an aspect, a disclosed linker can comprise the sequence of SEQ ID NO: 96. In an aspect, a disclosed linker can be a rigid linker.

[0106] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein is a recombinant enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity.

[0107] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%. at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NQ:20 or SEQ ID NO:21 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21 and having IgM specific protease activity’ and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%. at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity.

[0108] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23. Disclosed herein is a recombinant enzyme com rising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity.

[0109] Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO:24. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO:27. Disclosed herein is an enzy me comprising the sequence set forth in SEQ ID NO:30. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO:33. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO: 36.

[01 10] Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO:39. Disclosed herein is an enzyme comprising the sequence set forth in SEQ ID NO:42.

[0111] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%. at least 70%. at least 80%. or at least 90% identity to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%. at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26 or a fragment thereof and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26 or a fragment thereof and having IgM specific protease activity.

[0112] Disclosed herein is a recombinant enzy me comprising the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%. at least 60%. at least 70%. at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a fragment thereof. Disclosed herein is a recombinant enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%. at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a fragment thereof and having IgM specific protease activity⁷. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a fragment thereof and having IgM specific protease activity.

[01 13] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%. at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO: 31 or SEQ ID NO:32 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32 or a fragment thereof and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32 or a fragment thereof and having IgM specific protease activity.

[0114] Disclosed herein is a recombinant enzy me comprising the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%. at least 60%. at least 70%. at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35 or a fragment thereof. Disclosed herein is a recombinant enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%. at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO: 34 or SEQ ID NO:35 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35 or a fragment thereof and having IgM specific protease activity⁷. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35 or a fragment thereof and having IgM specific protease activity.

[01 15] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%. at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO: 37 or SEQ IDNO:38 and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38 or a fragment thereof and having IgM specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38 or a fragment thereof and having IgM specific protease activity.

[0116] Disclosed herein is a recombinant enzy me comprising the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%. at least 60%. at least 70%. at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41 or a fragment thereof. Disclosed herein is a recombinant enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity.

[0117] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity’ to the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44 and having IgM specific protease activity and IgG specific protease activity⁷. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity.

[0118] Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46 or a fragment thereof. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity.

[0119] In an aspect, a disclosed recombinant enzy me can have IgM specific protease activity'. In an aspect, a disclosed recombinant enzyme can cleave the IgM the heavy chain at the interdomain region between constant domains CLL2 and Cp3. In an aspect, a disclosed recombinant enzyme can cleave human IgM. In an aspect, a disclosed recombinant enzyme can cleave non-human primate IgM. In an aspect, a disclosed recombinant enzy me does not cleave non-human IgM. For example, in an aspect, a disclosed recombinant enzyme does not cleave dog IgM, pig IgM, and mouse IgM. In an aspect, a disclosed IgM specific recombinant enzyme does not cleave human IgA, IgD, IgE, IgG, or any combination thereof.

[0120] In an aspect, a disclosed recombinant enzyme can have a binding domain specific for IgM. In an aspect, a disclosed recombinant enzy me can cleave IgM between position 350 and position

IgM (for example, the substrate). In an aspect, a disclosed recombinant enzyme can have a binding domain capable of specifically binding to one or more amino acid residues 355 through 360 of IgM. In an aspect, a disclosed recombinant enzyme can have an affinity for human IgM. In an aspect, a disclosed IgM can be cleaved at SEQ ID NO: 110. In an aspect, a disclosed IgM can be cleaved at the sequence comprising 355D, 356T. 357A, 3581, 359R. and 360V. In an aspect, a disclosed IgM can be cleaved at position 355 through position 360 of SEQ ID NO: 105.

[0121] In an aspect, a disclosed recombinant enzyme can have IgG and IgM specific protease activity. In an aspect, a disclosed recombinant enzyme can cleave the IgM the heavy chain at the interdomain region between constant domains Cp2 and Cp3 and can cleave the IgG below the hinge region. In an aspect, a disclosed recombinant enzyme can cleave the IgG below the hinge region, thereby yielding Ftab'h and Fc fragments. In an aspect, a disclosed recombinant enzyme can cleave the IgG separating the Cp2 and Cp3 domains from the Cpl domain.

[0122] In an aspect, a disclosed recombinant enzyme can cleave human IgG and human IgM. In an aspect, a disclosed recombinant enz me can cleave non-human primate IgG and non-human primate IgM. In an aspect, a disclosed recombinant enzyme having IgG and IgM protease activity does not cleave IgA, IgD, IgE, or any combination thereof. In an aspect, a disclosed recombinant enzyme having IgG and IgM protease activity does not cleave non-human IgM or non-human IgG. For example, in an aspect, a disclosed recombinant enzyme having IgG and IgM protease activity does not cleave dog IgM, pig IgM, and mouse IgM, or dog IgG, pig IgG, or mouse IgG.

[0123] In an aspect, a disclosed recombinant enzyme can have a binding domain capable of specifically binding to the sequence of SEQ ID NO: 111, SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO: 114, or any combination thereof. In an aspect, a disclosed recombinant enzyme can have a binding domain capable of specifically binding to the sequence of SEQ ID NO: 1 15, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof. In an aspect, a disclosed recombinant enzyme can cleave IgG at amino acid residues 109 through 124 of the IgG of SEQ ID NO: 106, residues 106 through 120 of the IgG of SEQ ID NO: 107, residues 156 through 171 of the IgG of SEQ ID NO: 108. residues 106 through 121 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, a disclosed recombinant enzyme can cleave IgG at amino acid residues 116 through 122 of the IgG of SEQ ID NO: 106, residues 112 through 118 of the IgG of SEQ ID NO: 107, residues 163 through 169 of the IgG of SEQ ID NO: 108, residues 113 through 119 oflgG of SEQ ID NO: 109, or any combination thereof. In an aspect, a disclosed recombinant enzyme can cleave one or more IgG molecules at SEQ ID NO: 115, SEQ ID NO: 1 16, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof.

[0124] In an aspect, a disclosed recombinant enzy me can be specific for IgM and IgG, can cleave IgM between position 355 and position 360 of SEQ ID NO: 105, and can cleave at residues 116 through 122 of the IgG of SEQ ID NO: 106, residues 112 through 118 of the IgG of SEQ ID NO: 107, residues 163 through 169 of the IgG of SEQ ID NO: 108, and residues 113 through 119 of IgG of SEQ ID NO: 109. In an aspect, a disclosed recombinant enzyme can be specific for IgM and IgG can recognize and cleave SEQ ID NO: 110 in IgM and cleave SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117. SEQ ID NO: 118 in IgG

[0125] In an aspect, a disclosed recombinant enzyme can clear and/or remove IgM and/or IgM and IgG from circulation in a subject. In an aspect, a disclosed recombinant enzyme can clear and/or remove IgM and/or IgM and IgG from circulation in the sera of a subject. In an aspect, a disclosed recombinant enzyme can cleave and/or degrade circulating IgM and/or IgM and IgG in a subject. In an aspect, a disclosed recombinant enzyme can transiently clear and/or remove IgM and/or IgM and IgG from circulation in the sera of a subject, or can transiently cleave and/or degrade circulating IgM and/or IgM and IgG in a subject. In an aspect, a disclosed recombinant enzyme can reduce and/or diminish the level of circulating IgM and/or IgG and IgM levels in a subject, or can transiently reduce and/or diminish the level of circulating IgM and/or IgG and IgM levels in a subject. In aspect, a disclosed transient reduction and/or diminishment can comprise about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[0126] In an aspect, a disclosed recombinant enzyme can block and/or minimize activation of the complement cascade. In an aspect, a disclosed recombinant enzyme can block and/or minimize activation of the formation of the Cl complex and/or C3 complex. In an aspect, a disclosed recombinant enzyme can block and/or minimize activation of the classical complement pathway. In an aspect, a disclosed recombinant enzyme can transiently block and/or minimize activation of the complement cascade, can transiently block and/or minimize activation of the formation of the Cl complex and/or C3 complex, can transiently block and/or minimize activation of the classical complement pathway, or any combination thereof.

[0127] In an aspect, a disclosed recombinant enzyme can remove and/or cleave surface-bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells. In an aspect, the disclosed removal and/or cleavage of IgM from the surface of B cells can be reversible and/or transient. In an aspect, a disclosed recombinant enzyme can transiently remove and/or cleave surface-bound IgM from B cells and/or can transiently remove and/or cleave IgM from the surface of B cells. In an aspect, a disclosed recombinant enzyme can remove and/or cleave surface bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells without killing and/or injuring the B cells. [0128] In an aspect, a disclosed recombinant enzyme can reduce and/or minimize vector mediated immunotoxicity in a subject. In an aspect, a disclosed recombinant enzyme can enable and/or allow vector re-dosing in a subject. In an aspect, a disclosed recombinant enzyme can reduce and/or minimize transgene immunogenicity in a subject.

[0129] In an aspect, a disclosed recombinant enzyme can improve and/or enhance transgene efficacy and/or expression in a subj ect. In an aspect, a disclosed recombinant enzyme can improve and/or enhance transgene efficacy and/or expression in a subject. In an aspect, improving and/or enhancing transgene efficacy and/or expression in a subject can comprise a 10%, 20%, 30%, 40%, 50%. 60%. 70%. 80%. 90%. 100%, or any amount of an improvement and/or enhancement when compared to a pre-existing level of transgene efficacy and/or expression such as, for example, a pre-treatment level (prior to administering one or more disclosed recombinant enzymes). In an aspect, the amount of improvement and/or enhancement can be 10-20%, 20-30%, 30-40%, 40- 50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as a pre-treatment level (prior to administering one or more disclosed recombinant enzymes). In an aspect, an improvement and/or enhancement can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having received or not having been administered one or more disclosed recombinant enzymes). In an aspect, an improvement and/or enhancement can be a partial or incomplete restoration. In an aspect, an improvement and/or enhancement can be complete or near complete such that the level of neutralizing antibodies or neutralizing IgG and/or IgM molecules is similar to that of a wild-type or control level.

[0130] In an aspect, a disclosed recombinant enzyme can decrease and/or minimize the severity’ of a rejection event of the one or more transplanted organs. In an aspect, a disclosed recombinant enzyme can prevent the rejection of the one or more transplanted organs. In an aspect, a disclosed rej ection event can be classified a hyperacute rej ection event, an acute rej ection event, or a chronic rejection event. In an aspect, following the administration and/or delivery of a disclosed recombinant enzyme, the one or more transplanted organs are not rejected by the subject. In an aspect, following the administration and/or delivery’ of a disclosed recombinant enzy me, the viability’ of one or more transplanted organs can be improved and/or increased. In an aspect, following the administration and/or delivery of a disclosed recombinant enzyme, the functionality' of one or more transplanted organs can be improved and/or increased. In an aspect, following the administration and/or delivery of a disclosed recombinant enzyme, the functionality of one or more transplanted organs is not decreased and/or diminished. In an aspect, a disclosed recombinant enzyme can degrade one or more anti-HLA antibodies directed at the one or more transplanted organs. [0131] In an aspect, a disclosed enzyme can be used to modulate humoral immunity in a subject. In an aspect, modulating humoral immunity in a subject can be an increase in the functionality of the subject’s humoral immunity, or can be a decrease in the functional of the subject's humoral immunity.

[0132] In an aspect, a disclosed enzyme can comprise one or more labels and/or tags. In an aspect, a disclosed label and/or a tag can comprise a fluorescent label and/or a tag. In an aspect, a disclosed label and/or a tag can comprise a carboxy -terminal label and/or tag or an amino-terminal label and/or tag. In an aspect, a disclosed label and/or a tag can comprise a carboxy -terminal fluorescent label and/or tag or an amino-terminal fluorescent label and/or tag. In an aspect, a disclosed label and/or tag can comprise an ALFA tag, an AviTag, a C tag, a Calmodulin-tag, an iCapTag (intein Capture Tag), a polyglutamate tag, a polyarginine tag, an E tag, a FLAG tag, a HA tag, a His tag, a Myc tag, a NE tag, a RholD4 tag, a S tag, a SBP tag, a Softag 1, a Softag 3, a Spot tag, a Strep tag, a T7 tag, a TC tag, a Ty tag, a V5 tag. a VSV-tag, an Xpress tag, an Isopeptag, a Spy tag, a SnoopTag, a SnoopTagJr, a DogTag, a SdyTag, a BCCP (Biotin Carboxyl Carrier Protein), a glutathione-S-transferase tag, a green fluorescent protein tag, a Halo tag, a SNAP -tag, a CLIP tag, a HUH tag, a maltose binding protein tag, a Nus tag, a thioredoxin tag, a Fc tag, a Designed Intrinsically Disordered tags containing disorder promoting amino acids, a Carbohydrate Recognition Domain or CRDSAT tag, a HiBiT-tag, or any combination thereof.

[0133] In an aspect, a disclosed label and/or a tag can comprise a fluorescent label or a fluorescent tag. In an aspect, a disclosed fluorescent label or disclosed fluorophore can comprise enhanced green fluorescent protein (EGFP), mEmerald, enhanced yellow fluorescent protein (EYFP), mApple, TdTomato, mCherry, miRFP670. any known fluorescent label or tag, or any combination thereof. In an aspect, a disclosed fluorescent label or disclosed fluorophore can comprise any fluorescent label or fluorophore that is amendable to analysis via flow cytometry. In an aspect, a pair of disclosed fluorescent labels or disclosed fluorophores can comprise any pair of fluorescent labels or fluorophores that is amendable to analysis via flow cytometry and have excitation and emission spectra that can be isolated or separated from each other, thereby enabling the interrogation of the docking interactions. Fluorophores and fluorescent labels are known in the art.

[0134] In an aspect, a disclosed enzy me or disclosed recombinant enzyme can be therapeutically effective when the dose comprises about 0.01 mg/kg body weight to about 100 mg/kg body w eight. In an aspect, a disclosed enzyme or disclosed recombinant enzyme can be therapeutically effective when the dose comprises about 0.01 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg body weight.

[0135] Disclosed herein is a recombinant enzy me comprising the sequence of SEQ ID NO:119. Disclosed herein is a recombinant enzy me encoded by the codon-optimized nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a recombinant enzyme encoded by a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity⁷ to the codon-optimized sequence of SEQ ID NO: 160 or SEQ ID NO: 161.

[0136] Disclosed herein is a recombinant enzyme comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein is a recombinant enzyme comprising the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 119. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity⁷ to the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159.

[0137] Disclosed herein is a recombinant enzyme comprising the sequence of SEQ ID NO: 119 and having human and non-human primate IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121 and having human and non-human primate IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159 and having human and non-human primate IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity⁷ to the sequence of SEQ ID NO: 119 and having human and non-human primate IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity⁷ to the sequence of SEQ ID NO: 120 or SEQ ID NO: 121 and having human and non-human primate IgG specific protease activity. Disclosed herein is a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159 and having human and non-human primate IgG specific protease activity.

[0138] Disclosed herein is a recombinant enzyme encoded by the codon-optimized nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161 and having human and non-human primate IgG specific protease activity. Disclosed herein is a recombinant enzyme encoded by a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the codon-optimized nucleic acid sequence of SEQ ID NO: 1 0 or SEQ ID NO: 161 and having human and non-human primate IgG specific protease activity.

2. Nucleic Acid Molecules

[0139] Disclosed herein is an isolated nucleic acid molecule encoding any disclosed enzyme. Disclosed herein is an isolated nucleic acid molecule encoding any disclosed recombinant enzyme. Disclosed herein is an isolated nucleic acid molecule comprising the sequence of any one of SEQ ID NO:47 - SEQ ID NO: 92.

[0140] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:01. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:24. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:47. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:70.

[0141] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:04. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:27. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:50. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:73.

[0142] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:07. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising the sequence set forth in SEQ ID NO:30. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:53. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:76.

[0143] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 10. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:33. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:56. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:79.

[0144] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 13. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:36. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:59. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:82.

[0145] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 16. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:39. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:62. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:85. [0146] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 19. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:42. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:65. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:88. [0147] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity' to the sequence set forth in SEQ ID NO: 02 or SEQ ID NO:03. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03 and having IgM specific protease activity'. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity' to the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03 or a fragment thereof and having IgM specific protease activity⁷. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03 or a fragment thereof and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:48 or SEQ ID NO: 49. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:71 or SEQ ID NO:72.

[0148] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%. at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06 and having IgM specific protease activity'. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity' to the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06 and having IgM specific protease activity⁷. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06 or a fragment thereof and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity' to the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06 or a fragment thereof and having IgM specific protease activity'. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:51 or SEQ ID NO: 52. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:74 or SEQ ID NO:75.

[0149] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the recombinant enzyme comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity⁷, or at least 90% identity⁷ to the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the recombinant enzyme comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09 or a fragment thereof and having IgM specific protease activity⁷. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%. at least 70%. at least 80%. or at least 90% identity to the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09 or a fragment thereof and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:54 or SEQ ID NO:55. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:77 or SEQ ID NO: 78.

[0150] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity⁷, or at least 90% identity to the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity⁷ to the sequence set forth in SEQ ID NO:11 or SEQ ID NO: 12 and having IgM specific protease activity⁷. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12 or a fragment thereof and having IgM specific protease activity⁷. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity⁷ to the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12 or a fragment thereof and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:57 or SEQ ID NO: 58. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 80 or SEQ ID NO: 81. [0151] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity’ to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity’ to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15 or a fragment thereof and having IgM specific protease activity⁷. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%. at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15 or a fragment thereof and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:60 or SEQ ID NO:61. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:83 or SEQ ID NO:84.

[0152] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%. at least 60%. at least 70%, at least 80% identity⁷, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:63 or SEQ ID NO:64. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:86 or SEQ ID NO:87.

[0153] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO: 20 or SEQ ID NO:21. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%. at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NQ:20 or SEQ ID NO:21 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:20 or SEQ ID NO:2I and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity⁷ to the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ IDNO:66 or SEQ ID NO:67. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 89 or SEQ ID NO:90.

[0154] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO: 22 or SEQ ID NO:23. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme compnsing the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme comprising the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:68 or SEQ ID NO:69. Disclosed herein is an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:91 or SEQ ID NO:92.

[0155] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:24. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:27. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:30. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me having the sequence set forth in SEQ ID NO:33. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:36.

[0156] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:39. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:42.

[0157] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%. at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%. at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having sequence having at least 50%, at least 60%. at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26 or a fragment thereof and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:25 or SEQ ID NO:26 or a fragment thereof and having IgM specific protease activity.

[0158] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me having a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity', or at least 90% identity to the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a fragment thereof and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a fragment thereof and having IgM specific protease activity.

[0159] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%. at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me having the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32 or a fragment thereof and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:31 or SEQ ID NO:32 or a fragment thereof and having IgM specific protease activity.

[0160] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me having a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity⁷, or at least 90% identity⁷ to the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me having a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35 or a fragment thereof and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me having a sequence having at least 50%, at least 60%, at least 70%. at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:34 or SEQ ID NO:35 or a fragment thereof and having IgM specific protease activity.

[0161] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me having the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%. at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38 and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me having the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38 or a fragment thereof and having IgM specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:37 or SEQ ID NO:38 or a fragment thereof and having IgM specific protease activity. [0162] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity’ to the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%. at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41 or a fragment thereof and having IgM specific protease activity' and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:40 or SEQ ID NO:41 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity.

[0163] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me having a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity⁷, or at least 90% identity⁷ to the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzy me having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity⁷ to the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44 and having IgM specific protease activity and IgG specific protease activity⁷. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity⁷, or at least 90% identity to the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%. at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:43 or SEQ ID NO:44 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity.

[0164] Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46. Disclosed herein is an isolated nucleic acid molecule encoding the enz me having a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46 or a fragment thereof. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%, at least 80% identity, or at least 90% identity to the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46 and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule encoding the enzyme having a sequence having at least 50%, at least 60%, at least 70%. at least 80%, or at least 90% identity to the sequence set forth in SEQ ID NO:45 or SEQ ID NO:46 or a fragment thereof and having IgM specific protease activity and IgG specific protease activity.

[0165] In an aspect, a disclosed isolated nucleic acid molecule can comprise the nucleic acid sequence for one or more labels and/or tags. In an aspect, a disclosed label and/or a tag can comprise a fluorescent label and/or a tag. In an aspect, a disclosed label and/or a tag can comprise a carboxy -terminal label and/or tag or an amino-terminal label and/or tag. In an aspect, a disclosed label and/or a tag can comprise a carboxy-terminal fluorescent label and/or tag or an aminoterminal fluorescent label and/or tag. [0166] In an aspect, a disclosed label and/or tag can comprise an ALFA tag, an AviTag, a C tag, a Calmodulin-tag, an iCapTag (intein Capture Tag), a poly glutamate tag, a polyarginine tag, an E tag, a FLAG tag, a HA tag, a His tag, a Myc tag, a NE tag, a RholD4 tag, a S tag, a SBP tag, a Softag 1, a Softag 3, a Spot tag, a Strep tag, a T7 tag, a TC tag, a Ty tag, a V5 tag, a VSV-tag, an Xpress tag. an Isopeptag, a Spy tag. a SnoopTag. a SnoopTagJr. a DogTag. a SdyTag, a BCCP (Biotin Carboxyl Carrier Protein), a glutathione-S-transferase tag, a green fluorescent protein tag, a Halo tag, a SNAP-tag, a CLIP tag, a HUH tag, a maltose binding protein tag, a Nus tag, a thioredoxin tag, a Fc tag, a Designed Intrinsically Disordered tags containing disorder promoting amino acids, a Carbohydrate Recognition Domain or CRDSAT tag, a HiBiT-tag. or any combination thereof.

[0167] In an aspect, a disclosed label and/or a tag can comprise a fluorescent label or a fluorescent tag. In an aspect, a disclosed fluorescent label or disclosed fluorophore can comprise enhanced green fluorescent protein (EGFP), mEmerald. enhanced yellow fluorescent protein (EYFP), mApple, TdTomato, mCherry, miRFP670, any known fluorescent label or tag, or any combination thereof. In an aspect, a disclosed fluorescent label or disclosed fluorophore can comprise any fluorescent label or fluorophore that is amendable to analysis via flow cytometry⁷. In an aspect, a pair of disclosed fluorescent labels or disclosed fluorophores can comprise any pair of fluorescent labels or fluorophores that is amendable to analysis via flow cytometry and have excitation and emission spectra that can be isolated or separated from each other, thereby enabling the interrogation of interactions. Fluorophores and fluorescent labels are known in the art.

[0168] In an aspect of a disclosed nucleic acid molecule, a disclosed encoded recombinant enzyme can have IgM specific protease activity. In an aspect, a disclosed encoded recombinant enzyme can cleave the IgM the heavy chain at the interdomain region between constant domains Cp2 and Cp3. In an aspect, a disclosed encoded recombinant enzyme can cleave human IgM. In an aspect, a disclosed encoded enzyme can cleave non-human primate IgM. In an aspect, a disclosed encoded recombinant enzyme does not cleave non-human IgM. For example, in an aspect a disclosed encoded recombinant enzyme does not cleave dog IgM, pig IgM, and mouse IgM. In an aspect, a disclosed encoded IgM specific recombinant enzyme does not cleave human IgA, IgD, IgE, IgG, or any combination thereof.

[0169] In an aspect, a disclosed encoded recombinant enzy me can have a binding domain specific for IgM. In an aspect, a disclosed encoded recombinant enzyme can cleave IgM between position 350 and position 365 of SEQ ID NO: 105. In an aspect, SEQ ID NO: 105 can represent the linear sequence of an IgM (for example, the substrate). In an aspect, a disclosed encoded recombinant enzy me can have a binding domain capable of specifically binding to one or more amino acid residues 355 through 360 of IgM. In an aspect, a disclosed encoded recombinant enzyme can have an affinity for human IgM. In an aspect, a disclosed IgM can be cleaved at SEQ ID NO: 110. In an aspect, a disclosed IgM can be cleaved at the sequence comprising 355D, 356T, 357A, 3581, 359R, and 360V. In an aspect, a disclosed IgM can be cleaved at position 355 through position 360 of SEQ ID NO: 105.

[0170] In an aspect, a disclosed encoded recombinant enzyme can have IgG and IgM specific protease activity. In an aspect, a disclosed encoded recombinant enzyme can cleave the IgM the heavy chain at the interdomain region between constant domains Cp2 and Cp3 and can cleave the IgG below the hinge region. In an aspect, a disclosed encoded recombinant enzyme can cleave the IgG below the hinge region, thereby yielding Ftab’fi and Fc fragments. In an aspect, a disclosed encoded recombinant enzyme can cleave the IgG separating the Cp2 and Cp3 domains from the Cpl domain.

[0171] In an aspect, a disclosed encoded recombinant enzyme can cleave human IgG and human IgM. In an aspect, a disclosed encoded recombinant enzyme can cleave non-human primate IgG and non-human primate IgM. In an aspect, a disclosed encoded recombinant enzyme having IgG and IgM protease activity does not cleave IgA, IgD, IgE, or any combination thereof. In an aspect, a disclosed encoded recombinant enzyme having IgG and IgM protease activity does not cleave non-human IgM or non-human IgG. For example, in an aspect, a disclosed encoded recombinant enzyme having IgG and IgM protease activity does not cleave dog IgM, pig IgM, and mouse IgM, or dog IgG, pig IgG, or mouse IgG.

[0172] In an aspect, a disclosed encoded recombinant enzyme can have a binding domain capable of specifically binding to the sequence of SEQ ID NO:111, SEQ ID NO: 112, SEQ ID NO:113, SEQ ID NO: 114, or any combination thereof. In an aspect, a disclosed encoded recombinant enzyme can have a binding domain capable of specifically binding to the sequence of SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof. In an aspect, a disclosed encoded recombinant enzy me can cleave IgG at amino acid residues 109 through 124 of the IgG of SEQ ID NO: 106, residues 106 through 120 of the IgG of SEQ ID NO: 107, residues 156 through 171 of the IgG of SEQ ID NO: 108, residues 106 through 121 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, a disclosed encoded recombinant enzyme can cleave IgG at amino acid residues 116 through 122 of the IgG of SEQ ID NO: 106, residues 112 through 118 of the IgG of SEQ ID NO: 107, residues 163 through 169 of the IgG of SEQ ID NO: 108. residues 113 through 119 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, a disclosed encoded recombinant enzyme can cleave one or more IgG molecules at SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof. [0173] In an aspect, a disclosed encoded recombinant enzyme can be specific for IgM and IgG, can cleave IgM between position 355 and position 360 of SEQ ID NO: 105, and can cleave at residues 116 through 122 of the IgG of SEQ ID NO: 106, residues 112 through 118 of the IgG of SEQ ID NO: 107, residues 163 through 169 of the IgG of SEQ ID NO: 108, and residues 113 through 119 of IgG of SEQ ID NO: 109. In an aspect, a disclosed encoded recombinant enzyme can be specific for IgM and IgG can recognize and cleave SEQ ID NO: 1 10 in IgM and cleave SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118 m IgG

[0174] In an aspect, a disclosed encoded recombinant enzyme can clear and/or remove IgM and/or IgM and IgG from circulation in a subject. In an aspect, a disclosed recombinant enzyme can clear and/or remove IgM and/or IgM and IgG from circulation in the sera of a subject. In an aspect, a disclosed recombinant enzyme can cleave and/or degrade circulating IgM and/or IgM and IgG in a subject. In an aspect, a disclosed recombinant enzyme can transiently clear and/or remove IgM and/or IgM and IgG from circulation in the sera of a subject, or can transiently cleave and/or degrade circulating IgM and/or IgM and IgG in a subject. In an aspect, a disclosed recombinant enzyme can reduce and/or diminish the level of circulating IgM and/or IgM and IgG levels in a subject, or can transiently reduce and/or diminish the level of circulating IgM and/or IgM and IgG levels in a subject. In aspect, a disclosed transient reduction and/or diminishment can comprise about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[0175] In an aspect of a disclosed nucleic acid molecule, a disclosed encoded enzy me can block and/or minimize activation of the complement cascade. In an aspect, a disclosed encoded enzyme can block and/or minimize activation of the formation of the Cl complex and/or C3 complex. In an aspect, a disclosed encoded enzyme can block and/or minimize activation of the classical complement pathway. In an aspect, a disclosed encoded enz me can transiently block and/or minimize activation of the complement cascade, can transiently block and/or minimize activation of the formation of the Cl complex and/or C3 complex, can transiently block and/or minimize activation of the classical complement pathway, or any combination thereof.

[0176] In an aspect of a disclosed nucleic acid molecule, a disclosed encoded enzyme can remove and/or cleave surface-bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells. In an aspect, the disclosed removal and/or cleavage of IgM from the surface of B cells can be reversible and/or transient. In an aspect, a disclosed encoded enzyme can transiently remove and/or cleave surface-bound IgM from B cells and/or can transiently remove and/or cleave IgM from the surface of B cells. In an aspect, a disclosed encoded enzyme can remove and/or cleave surface bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells without killing and/or injuring the B cells.

[0177] In an aspect of a disclosed nucleic acid molecule, a disclosed encoded enzyme can reduce and/or minimize vector mediated immunotoxicity in a subject. In an aspect of a disclosed nucleic acid molecule, a disclosed encoded enzyme can enable and/or allow vector re-dosing in a subject. In an aspect of a disclosed nucleic acid molecule, a disclosed encoded enzyme can reduce and/or minimize transgene immunogenicity in a subject. In an aspect of a disclosed nucleic acid molecule, a disclosed encoded enzyme can improve and/or enhance transgene efficacy and/or expression in a subject.

[0178] In an aspect, a disclosed nucleic acid molecule can improve and/or enhance transgene efficacy and/or expression in a subject. In an aspect, a disclosed nucleic acid molecule can improve and/or enhance transgene efficacy and/or expression in a subject. In an aspect, improving and/or enhancing transgene efficacy and/or expression in a subject can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of an improvement and/or enhancement when compared to a pre-existing level of transgene efficacy and/or expression such as, for example, a pre-treatment level (prior to administering one or more a disclosed nucleic acid molecules). In an aspect, the amount of improvement and/or enhancement can be 10-20%, 20- 30%. 30-40%, 40-50%, 50-60%. 60-70%, 70-80%, 80-90%. or 90-100% more than a pre-existing level such as a pre-treatment level (prior to administering one or more a disclosed nucleic acid molecule). In an aspect, an improvement and/or enhancement can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having received or not having been administered one or more a disclosed nucleic acid molecules). In an aspect, an improvement and/or enhancement can be a partial or incomplete restoration. In an aspect, an improvement and/or enhancement can be complete or near complete such that the level of neutralizing antibodies or neutralizing IgG and/or IgM molecules is similar to that of a wildtype or control level.

[0179] In an aspect, a disclosed nucleic acid molecule and/or a disclosed nucleic acid sequence can be CpG depleted and codon-optimized for expression in a human cell. In an aspect, “CpG- free” can mean completely free of CpGs or partially free of CpGs. In an aspect, “CpG-free” can mean “CpG-depleted”. In an aspect, “CpG-depleted” can mean “CpG-free'’. In an aspect, “CpG- depleted” can mean completely depleted of CpGs or partially depleted of CpGs. In an aspect, “CpG-free” can mean “CpG-optimized” for a desired and/or ideal expression level. CpG depletion and/or optimization is know n to the skilled person in the art.

[0180] In an aspect, a “codon-optimized” gene can be a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell (i.e.. a human cell or a human subject). In an aspect, a disclosed nucleic acid molecule and/or a disclosed nucleic acid sequence can be codon-optimized, either wholly or in part. Because any one amino acid (except for methionine and tryptophan) is encoded by several codons, the sequence of the nucleic acid molecule can be changed without changing the encoded amino acid. In an aspect, a disclosed nucleic acid sequence can be codon-optimized for expression in a human cell or a human organ or one or more human cells or one or more human organs. In an aspect, a disclosed nucleic acid sequence can be codon-optimized for expression in a human subject.

[0181] In an aspect, a disclosed codon-optimized nucleic acid sequence can comprise SEQ ID NO:48. SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO : 74, SEQ ID NO : 75 , SEQ ID NO : 77, SEQ ID NO : 78, SEQ ID NO : 80, SEQ ID NO : 81 , SEQ ID NO: 83, SEQ ID NO:84, SEQ ID NO: 86. SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, or any combination thereof.

[0182] In an aspect, a disclosed nucleic acid sequence can encode the protein or a portion thereof (such as, for example, Exon 1 or Exon 4, etc.) associated with the following genes: ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, AC AN, ADAMTS13, ADCY10, ADGRV1, AGL. AGRN, AHDC1, ALK, ALMS 1, ALPK3, ALS2, ANAPC 1, ANK.1, ANK2, ANKA, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR. BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTAI, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR/ABCC7, CHD1, CHD2, CHD3, CHD4. CHD7. CHD8, CIC, CIT. CLIP1, CLTC, CNOT1, CNTNAP1. COL11A1. COL11A2, COL12A1, COL17A1, COL18A1, COL1A1 , COL1 A2, COL27A1 , COL2A1 , COL3A1 , COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B, DLC1, DMD. DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, DNMBP, DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF, EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1, FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2, FYC01, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1, HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R, IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A, KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586. KIAA1109, KIAA1549, KIDINS220, KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1, LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, L0XHD1, LPA, LRBA, LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD, MAGI2, MAP1B, MAP3K1, MAPK8IP3. MAPKBPL MAST1, MBD5, MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO18B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN. NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF. NF1, NFASC, NHS, NIN, N1PBL, NLRP1, NOTCH1, NOTCH2. NOTCH3. NPHP4. NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205, OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO1, PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC. PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4. PRKDC. PRPF8, PRR12, PRX, PTCHI, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAIL RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS 1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A. SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJL SZT2. TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2. TECTA, TENM3, TENM4, TET3, TEX14, TEX15. TG, THOC2. TMEM94, TNC. TNIK, TNR, TNRC6B, TNXB, TOGARAMI, TONSL, TRIO, TRIOBP, TRIP1 1 , TRIP12, TRPM1 , TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26. ZNF142, ZNF292, ZNF335. ZNF407, ZNF462, ZNF469. or a portion thereof.

[0183] In an aspect, a disclosed nucleic acid sequence can encode a gene or a portion thereof (e.g., a specific exon such as an exon having a mutation) with a gene product that is directly or indirectly linked to one or more genetic diseases. Such genes include but are not limited to the following: dystrophin including mini- and micro-dystrophins (DMD); titin (TTN); titin cap (TCAP) a- sarcoglycan (SGCA), P-sarcoglycan (SGCB), y-sarcoglycan (SGCG) or 8-sarcoglycan (SGCD); alpha- 1 -antitrypsin (Al-AT); myosin heavy chain 6 (MYH6); myosin heavy chain 7 (MYH7); myosin heavy chain 11 (MYH11); myosin light chain 2 (ML2); myosin light chain 3 (ML3); myosin light chain kinase 2 (MYLK2); myosin binding protein C (MYBPC3); desmin (DES); dynamin 2 (DNM2); laminin a2 (LAMA2); lamin A/C (LMNA); lamin B (LMNB); lamin B receptor (LBR); dysferlin (DYSF); emerin (EMD); insulin; blood clotting factors, including but not limited to, factor VIII and factor IX; erythropoietin (EPO); lipoprotein lipase (LPL); sarcoplasmic reticulum Ca2++-ATPase (SERCA2A), S100 calcium binding protein Al (SI 00 Al); myotubularin (MTM); DM1 protein kinase (DMPK); glycogen phosphorylase L (PYGL); glycogen phosphorylase, muscle associated (PYGM); glycogen synthase 1 (GYS I); glycogen synthase 2 (GYS2); a-galactosidase A (GLA); a-N-acetylgalactosaminidase (NAGA); acid a-glucosidase (GAA), sphingomyelinase phosphodiesterase 1 (SMPD1); lysosomal acid lipase (LIPA); collagen type I al chain (COL1A1); collagen type I a2 chain (COL1 A2); collagen type III al chain (COL3A1); collagen type V al chain (COL5A1); collagen type V a2 chain (COL5A2); collagen type VI al chain (COL6A1); collagen type VI a2 chain (COL6A2); collagen type VI a3 chain (COL6A3); procollagen-lysine 2-oxoglutarate 5-dioxygenase (PLOD1); lysosomal acid lipase (LIPA); frataxin (FXN); myostatin (MSTN); P-N-acetyl hexosaminidase A (HEXA); P-N-acetylhexosaminidase B (HEXB); P-glucocerebrosidase (GBA); adenosine monophosphate deaminase 1 (AMPD1); P-globin (HBB); iduronidase (IDUA); iduronate 2- sulfate (IDS); troponin 1 (TNNI3); troponin T2 (TNNT2); troponin C (TNNC1); tropomyosin 1 (TPM1); tropomyosin 3 (TPM3); N-acetyl-a-glucosaminidase (NAGLU); N-sulfoglucosamine sulfohydrolase (SGSH); heparan-a-glucosaminide N-acetyltransferase (HGSNAT); integrin a 7 (IGTA7); integrin a 9 (IGTA9); glucosamine(N-acetyl)-6-sulfatase (GNS); galactosamine(N- acetyl)-6-sulfatase (GALNS); P-galactosidase (GLB1); P-glucuronidase (GUSB); hyaluronoglucosaminidase 1 (HYAL1); acid ceramidase (ASAHI); galactosylcermidase (GALC); cathepsin A (CTSA); cathepsin D (CTSA); cathepsin K (CTSK); GM2 ganglioside activator (GM2A); arylsulfatase A (ARSA); arylsulfatase B (ARSB); formylglycine-generating enzyme (SUMFI); neuraminidase 1 (NEU1); N-acetylglucosamine-1 -phosphate transferase a (GNPTA); N-acetylglucosamine-1 -phosphate transferase P (GNPTB); N-acetylglucosamine-l-phosphate transferase y (GNPTG); mucolipin-1 (MCOLN1); NPC intracellular transporter 1 (NPC1); NPC intracellular transporter 2 (NPC2); ceroid lipofuscinosis 5 (CLN5); ceroid lipofuscinosis 6 (CLN6); ceroid lipofuscinosis 8 (CLN8); palmitoyl protein thioesterase 1 (PPT1); tripeptidyl peptidase 1 (TPP1); battenin (CLN3); DNAJheat shock protein family 40 member C5 (DNAJC5); major facilitator superfamily domain containing 8 (MFSD8); mannosidase a class 2B member 1 (MAN2B 1); mannosidase R (MANBA); aspartylglucosaminidase (AGA); a-L-fucosidase (FUCA1); cystinosin, lysosomal cysteine transporter (CTNS); sialin; solute carrier family 2 member 10 (SLC2A10); solute carrier family 17 member 5 (SLC17A5); solute carrier family 6 member 19 (SLC6A19); solute carrier family 22 member 5 (SLC22A5); solute carrier family 37 member 4 (SLC37A4); lysosomal associated membrane protein 2 (LAMP2); sodium voltagegated channel a subunit 4 (SCN4A); sodium voltage-gated channel P subunit 4 (SCN4B); sodium voltage-gated channel a subunit 5 (SCN5A); sodium voltage-gated channel a subunit 4 (SCN4A); calcium voltage-gated channel subunit ale (CACNA1C); calcium voltage-gated channel subunit als (CACNA1S); phosphoglycerate kinase 1 (PGK1); phosphoglycerate mutase 2 (PGAM2); amylo-a-l,6-glucosidase,4-a-glucanotransferase (AGL); potassium voltage-gated channel ISK- related subfamily member 1 (KCNE1); potassium voltage-gated channel ISK-related subfamily member 2 (KCNE2); potassium voltage-gated channel subfamily J member 2 (KCNJ2); potassium voltage-gated channel subfamily J member 5 (KCNJ5); potassium voltage-gated channel subfamily H member 2 (KCNH2); potassium voltage-gated channel KQT-like subfamily member 1 (KCNQ1); hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4); chloride voltage-gated channel 1 (CLCN1); carnitine palmitoyltransferase 1A (CPT1A); ryanodine receptor 1 (RYR1); ryanodine receptor 2 (RYR2); bridging integrator 1 (BINI); LARGE xylosyl- and glucuronyltransferase 1 (LARGE1); docking protein 7 (D0K7); fukutin (FKTN); fukutin related protein (FKRP); selenoprotein N (SELENON); protein O- mannosyltransferase 1 (POMT1); protein O-mannosyltransferase 2 (POMT2); protein O-linked mannose N-acetylglucosaminyltransferase 1 (POMGNT1); protein O-linked mannose N- acetylglucosaminyltransferase 2 (POMGNT2); protein-O-mannose kinase (POMK); isoprenoid synthase domain containing (ISPD); plectin (PLEC); cholinergic receptor nicotinic epsilon subunit (CHRNE); choline O-acetyltransferase (CHAT); choline kinase |3 (CHKB); collagen like tail subunit of asymmetric acetylcholinesterase (COLQ); receptor associated protein of the synapse (RAPSN); four and a half LIM domains 1 (FHL1); [3- 1 A-glucuron l transferase 1 (B4GAT1); P-l,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2); dystroglycan 1 (DAGI); transmembrane protein 5 (TMEM5); transmembrane protein 43 (TMEM43); SECIS binding protein 2 (SECISBP2); glucosamine (UDP-N-acety l)-2-epimerase/N-acety lmannosamine kinase (GNE); anoctamin 5 (ANO5); structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1); lactate dehydrogenase A (LDHA); lactate dehydrogenase B (LHDB); calpain 3 (CAPN3); caveolin 3 (CAV3); tripartite motif containing 32 (TRIM32); CCHC-type zinc finger nucleic acid binding protein (CNBP); nebulin (NEB); actin, al, skeletal muscle (ACTA1); actin, al, cardiac muscle (ACTC1); actinin a2 (ACTN2); poly(A)-binding protein nuclear 1 (PABPN1); LEM domain-containing protein 3 (LEMD3); zinc metalloproteinase STE24 (ZMPSTE24); microsomal triglyceride transfer protein (MTTP); cholinergic receptor nicotinic al subunit (CHRNA1); cholinergic receptor nicotinic a2 subunit (CHRNA2); cholinergic receptor nicotinic a3 subunit (CHRNA3); cholinergic receptor nicotinic a4 subunit (CEIRNA4); cholinergic receptor nicotinic a5 subunit (CHRNA5); cholinergic receptor nicotinic a6 subunit (CHRNA6); cholinergic receptor nicotinic a7 subunit (CHRN A7); cholinergic receptor nicotinic a8 subunit (CHRNA8); cholinergic receptor nicotinic a9 subunit (CHRNA9); cholinergic receptor nicotinic alO subunit (CHRNA10); cholinergic receptor nicotinic pi subunit (CHRNB1); cholinergic receptor nicotinic P2 subunit (CHRNB2); cholinergic receptor nicotinic P3 subunit (CHRNB3); cholinergic receptor nicotinic P4 subunit (CHRNB4); cholinergic receptor nicotinic y subunit (CHRNG1); cholinergic receptor nicotinic a subunit (CHRND); cholinergic receptor nicotinic E subunit (CHRNE1); ATP binding cassette subfamily A member 1 (ABCA1); ATP binding cassette subfamily C member 6 (ABCC6); ATP binding cassette subfamily C member 9 (ABCC9); ATP binding cassette subfamily D member 1 (ABCD1); ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 1 (ATP2A1); ATM serine/threonine kinase (ATM); a tocopherol transferase protein (TTP A); kinesin family member 21 A (KIF21A); paired-like homeobox 2a (PH0X2A); heparan sulfate proteoglycan 2 (HSPG2); stromal interaction molecule 1 (STIM1); notch 1 (NOTCHI); notch 3 (N0TCH3); dystrobrevin a (DTNA); protein kinase AMP-activated, noncatalytic y2 (PRKAG2); cysteine- and glycine-rich protein 3 (CSRP3); viniculin (VCL); myozenin 2 (MyoZ2); myopalladin (MYPN); junctophilin 2 (JPH2); phospholamban (PLN); calreticulin 3 (CALR3); nexilin F-actin-binding protein (NEXN); LIM domain binding 3 (LDB3); eyes absent 4 (EYA4); huntingtin (HTT); androgen receptor (AR); protein tyrosine phosphate non-receptor type 11 (PTPN11); junction plakoglobin (JUP); desmoplakin (DSP); plakophilin 2 (PKP2); desmoglein 2 (DSG2); desmocollin 2 (DSC2); catenin a3 (CTNNA3); NK2 homeobox 5 (NKX2-5); A-kinase anchor protein 9 (AKAP9); A-kinase anchor protein 10 (AKAP10); guanine nucleotide-binding protein a-inhibiting activity polypeptide 2 (GNAI2); ankyrin 2 (ANK2); syntrophin a-1 (SNTAT); calmodulin 1 (CALM1); calmodulin 2 (CALM2); HTRA serine peptidase 1 (HTRA1); fibrillin 1 (FBN1); fibrillin 2 (FBN2); xylosyltransferase 1 (XYLT1); xylosyltransferase 2 (XYLT2); tafazzin (TAZ); homogentisate 1 ,2-dioxygenase (HGD); glucose-6-phosphatase catalytic subunit (G6PC); 1,4- alpha-glucan enzyme 1 (GBE1); phosphofructokinase, muscle (PFKM); phosphorylase kinase regulatory subunit alpha 1 (PHKA1); phosphorylase kinase regulatory subunit alpha 2 (PHKA2); phosphorylase kinase regulator}' subunit beta (PHKB); phosphorylase kinase catalytic subunit gamma 2 (PHKG2); phosphoglycerate mutase 2 (PGAM2); cystathionine-beta-synthase (CBS); methylenetetrahydrofolate reductase (MTHFR); 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR); 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR); methylmalonic aciduria and homocystinuria, cblD type (MMADHC); mitochondrial DNA, including, but not limited to mitochondrially encoded NADH: ubiquinone oxidoreductase core subunit 1 (MT-ND1); mitochondrially encoded NADH: ubiquinone oxidoreductase core subunit 5 (MT-ND5); mitochondrially encoded tRNA glutamic acid (MT-TE); mitochondrially encoded tRNA histadine (MT-TH); mitochondrially encoded tRNA leucine 1 (MT-TL1); mitochondrially encoded tRNA lysine (MT-TK); mitochondrially encoded tRNA serine 1 (MT- TS1); mitochondrially encoded tRNA valine (MT-TV); mitogen-activated protein kinase 1 (MAP2K1); B-Raf proto-oncogene, serine/threonine kinase (BRAF); raf-1 proto-oncogene, serine/threonine kinase (RAFI); growth factors, including, but not limited to insulin growth factor 1 (IGF-1); transforming growth factor 03 (TGF03); transforming growth factor 0 receptor, type I (TGF0R1); transforming growth factor 0 receptor, type II (TGF0R2), fibroblast growth factor 2 (FGF2), fibroblast growth factor 4 (FGF4), vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B): vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), vascular endothelial growth factor receptor 1 (VEGFR1), and vascular endothelial growth factor receptor 2 (VEGFR2), or any combination thereof.

[0184] In an aspect, a disclosed nucleic acid sequence can encode an RNA. In an aspect, a disclosed encoded RNA can comprise ribosomal RNA (rRNA), transfer RNA (tRNA), heterogeneous nuclear RNA (hnRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), micro RNA (miRNA), Piwd-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), singe guide RNA (sgRNA), non-coding RNA (ncRNA), long non-coding RNA (IncRNA), 7SL, Xist, short enhancer RNA (eRNA), circular RNA, intergenic RNA, or any combination thereof. In an aspect, a disclosed encoded RNA can comprise IncRNA, siRNA, shRNA, sgRNA, circular RNA, snoRNA, miRNA, or any combination thereof. [0185] In an aspect, a disclosed encoded RNA can comprise a functional non-coding RNA element. In an aspect, a disclosed nucleic acid molecule can comprise the sequence for one or more regulatory elements. In an aspect, a disclosed regulatory element can be a promoter, an enhancer, an internal ribosomal entry site (IRES), or another expression control element (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Regulatory elements can include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). In an aspect, a disclosed promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the w ild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, a disclosed promoter can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from anon-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed gene. In an aspect, a disclosed promoter can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art. In an aspect, a disclosed promoter can be any eukaryotic RNA polymerase II promoter.

[0186] In an aspect, a disclosed nucleic acid molecule can be in an expression cassette. Expression cassettes are know n to the art and can comprise a promoter sequence, an open reading frame (for the transgene of interest), and a 3’ UTR (typically containing a polyA in eukaryotes). [0187] In an aspect, a disclosed isolated nucleic acid molecule can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed isolated nucleic acid molecule can restore the functionality and/or structural integrity⁷ of a missing, deficient, and/or mutant protein or enzy me.

[0188] In an aspect, upon expression, a disclosed isolated nucleic acid molecule can decrease and/or minimize the severity' of a rejection event of the one or more transplanted organs. In an aspect, upon expression, a disclosed isolated nucleic acid molecule can prevent the rejection of the one or more transplanted organs. In an aspect, a disclosed rejection event can be classified a hyperacute rejection event, an acute rejection event, or a chronic rejection event. In an aspect, upon expression, a disclosed isolated nucleic acid molecule, the one or more transplanted organs are not rejected by' the subject. In an aspect, upon expression, a disclosed isolated nucleic acid molecule, the viability⁷ of one or more transplanted organs can be improved and/or increased. In an aspect, upon expression, a disclosed isolated nucleic acid molecule, the functionality of one or more transplanted organs can be improved and/or increased. In an aspect, upon expression, a disclosed isolated nucleic acid molecule, the functionality of one or more transplanted organs is not decreased and/or diminished. In an aspect, a disclosed encoded recombinant enzyme can degrade one or more anti-HLA antibodies directed at the one or more transplanted organs.

[0189] Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the recombinant enzyme of SEQ ID NO: 119. Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the recombinant enzyme of any⁷ one of SEQ ID NO: 120 - SEQ ID NO: 121. Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the recombinant enzyme of any one of SEQ ID NO: 122 - SEQ ID NO: 159. Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the recombinant enzyme of SEQ ID NO: 119, which has IgG specific protease activity. Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the recombinant enzy me of any one of SEQ ID NO: 120 - SEQ ID NO: 121, which has IgG specific protease activity’. Disclosed herein is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the recombinant enzyme of any one of SEQ ID NO: 122 - SEQ ID NO: 159, which has IgG specific protease activity⁷.

[0190] Disclosed herein is an isolated nucleic acid molecule encoding a recombinant enzyme comprising the nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a nucleic acid sequence encoding a recombinant enzyme and having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity⁷ to the nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a nucleic acid sequence encoding a recombinant enzyme having human and non-human primate IgG specific protease activity and having the nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a recombinant enzyme encoded by a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity⁷ to the codon-optimized nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161 and having human and non-human primate IgG specific protease activity.

[0191] Disclosed herein is an expression cassette comprising a disclosed nucleic acid sequence. Disclosed herein is an expression cassette comprising an isolated nucleic acid molecule encoding any disclosed enzyme. Disclosed herein is an expression cassette comprising an isolated nucleic acid molecule encoding any disclosed recombinant enzyme. Disclosed herein is an expression cassette comprising an isolated nucleic acid molecule comprising the sequence of any one of SEQ ID NO:47 - SEQ ID NO:92. Disclosed herein is an expression cassette comprising an isolated nucleic acid molecule comprising the sequence of SEQ ID NO: 160 or SEQ ID NO: 161.

3. Vectors

[0192] Disclosed herein is a vector comprising a disclosed isolated nucleic acid molecule. Disclosed herein is a vector comprising one or more disclosed isolated nucleic acid molecules. Disclosed herein is a vector comprising a disclosed isolated nucleic acid molecule have IgM specific protease activity⁷. Disclosed herein is a vector comprising a disclosed isolated nucleic acid molecule having IgM and IgG specific protease activity. Disclosed herein is a vector comprising a disclosed isolated nucleic acid molecule having IgM specific protease activity. Disclosed herein is a vector comprising a disclosed isolated nucleic acid molecule have IgM and IgG specific protease activity⁷. Disclosed herein is a vector comprising a nucleic acid sequence encoding the enzyme of any one of SEQ ID NO: 01 - SEQ ID NO: 46 or a fragment thereof. Disclosed herein is a vector comprising a nucleic acid sequence encoding the recombinant enzyme of any one of SEQ ID NO:02, SEQ ID NO:03, SEQ ID NO:05, SEQ ID NO:06, SEQ ID NO:08, SEQ ID NO: 09, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:21. SEQ ID NO:22, SEQ ID NO 23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29. SEQ ID NO:31, SEQ ID NO:32. SEQ ID NO:34, SEQ ID NO 35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO 41, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46. Disclosed herein is a vector comprising a nucleic acid sequence encoding the enzyme of any one of SEQ ID NO:01, SEQ ID NO:04, SEQ ID NO:07, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:39, and SEQ ID NO:42. Disclosed herein is a vector comprising a disclosed expression cassette.

[0193] Disclosed herein is a vector comprising a nucleic acid sequence comprising the sequence of any one of SEQ ID NO:47 - SEQ ID NO:92 or a fragment thereof. Disclosed herein is a vector comprising a nucleic acid sequence of any one of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO 52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO 58, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:72. SEQ ID NO:74, SEQ ID NO 75, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:81. SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, and SEQ ID NO:87. Disclosed herein is a vector comprising a nucleic acid sequence of any one of SEQ ID NO:01, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO 73, SEQ ID NO:76, SEQ ID NO:79, and SEQ ID NO:82.

[0194] Disclosed herein is a vector comprising a nucleic acid sequence comprising the sequence of any one of SEQ ID NO:47 - SEQ ID NO:92 or a fragment thereof. Disclosed herein is a vector comprising a nucleic acid sequence of any one of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55. SEQ ID NO:57, SEQ ID NO 58, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64. SEQ ID NO:66, SEQ ID NO:67. SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:71 , SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO 75, SEQ ID NO:77, SEQ ID NO : 78, SEQ ID NO : 80, SEQ ID NO : 81 , SEQ ID NO : 83, SEQ ID NO : 84, SEQ ID NO : 86, and SEQ ID NO:87. Disclosed herein is a vector comprising a nucleic acid sequence of any one of SEQ ID NO:01, SEQ ID NO:47. SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, and SEQ ID NO:82.

[0195] Disclosed herein is a vector comprising a nucleic acid sequence encoding the recombinant enzyme of SEQ ID NO: 119. Disclosed herein is a vector comprising a nucleic acid sequence encoding the recombinant enzyme of SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein is a vector comprising a nucleic acid sequence encoding the recombinant enzyme of any one of SEQ ID NO: 122 - SEQ ID NO: 159. Disclosed herein is a vector comprising a nucleic acid sequence encoding the recombinant enzyme of SEQ ID NO: 119 or a fragment thereof. Disclosed herein is a vector comprising a nucleic acid sequence encoding the recombinant enzyme of SEQ ID NO: 120 or SEQ ID NO: 121 or a fragment thereof. Disclosed herein is a vector comprising a nucleic acid sequence encoding the recombinant enzyme of any one of SEQ ID NO: 122 - SEQ ID NO: 159 or a fragment thereof.

[0196] Disclosed herein is a vector comprising a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a vector comprising a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161 or a fragment thereof.

[0197] In an aspect, a disclosed vector can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed vector can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0198] In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹⁰ vg/kg to about 2 x 10¹⁴ vg/kg. In an aspect, for example, a disclosed vector can be administered at a dose of about 1 x 10¹¹ to about 8 x 10¹³ vg/kg or about 1 x 10¹² to about 8 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 10¹³ to about 6 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of at least about 1 x 10¹⁰, at least about 5 x 10¹⁰. at least about 1 x 10¹¹, at least about 5 x 10¹¹, at least about 1 x 10¹², at least about 5 x 10¹², at least about 1 x 10¹³, at least about 5 x 10¹³, or at least about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered at a dose of no more than about 1 x 10¹⁰, no more than about 5 x 10¹⁰, no more than about 1 x 10¹¹, no more than about 5 x 10¹¹, no more than about 1 x 10¹², no more than about 5 x 10¹², no more than about 1 x 10¹³. no more than about 5 x 10¹³, or no more than about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 10¹² vg/kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 10¹¹ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total. In an aspect, a disclosed vector can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results. In an aspect, a therapeutically effective amount of disclosed vector can comprise a range determined by a skilled person. In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹² vg per subj ect total to about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total.

[01 9] In an aspect, a disclosed nucleic acid sequence can have a coding sequence that is less than about 4.5 kilobases.

[0200] In an aspect, a disclosed vector can be a viral vector or a non-viral vector. In an aspect, a disclosed non-viral vector can be a polymer-based vector, a peptide-based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid-based vector. In an aspect, a disclosed vector can comprise exosomes, extracellular vesicles, and virus like particles. In an aspect, a disclosed viral vector can be an adenovirus vector, an AAV vector, a herpes simplex virus vector, a retrovirus vector, a lentivirus vector, and alphavirus vector, a Flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease viral vector, a poxvirus vector, or a picomavirus vector.

[0201] In an aspect, a disclosed viral vector can be an adeno-associated virus (AAV) vector In an aspect, a disclosed AAV vector can include naturally isolated serotypes including, but not limited to, AAV1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAVrh39, AAVrh43, AAVcy.7 as well as bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, non-primate AAV, and any other virus classified by the International Committee on Taxonomy of Viruses (ICTV) as an AAV. In an aspect, an AAV capsid can be a chimera either created by capsid evolution or by rational capsid engineering from a naturally isolated AAV variants to capture desirable serotype features such as enhanced or specific tissue tropism and/or a host immune response escape. Naturally isolated AAV variants include, but not limited to, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV- 1829. AAV2 Y/F, AAV2 T/V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-Stnng (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81. In an aspect, a disclosed AAV vector can be AAV-Rh74 or a related variant (e.g., capsid variants like RHM4-1). In an aspect, a disclosed AAV vector can be AAV 8. In an aspect, a disclosed AAV vector can be AAVhum.8. In an aspect, a disclosed AAV vector can be a self-complementary AAV as disclosed herein.

[0202] In an aspect, a disclosed vector can comprise one or more promoters operably linked to a disclosed transgene and/or a disclosed nucleic acid sequence. In an aspect, a disclosed promoter can be positioned 5’ (upstream) or 3’ (downstream) of a disclosed transgene and/or a disclosed nucleic acid sequence under its control. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0203] In an aspect, a disclosed vector can further comprise one or more regulatory elements. In an aspect, a disclosed "‘regulatory element" can refer to promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Regulatory elements are discussed infra and can include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).

[0204] In an aspect, a disclosed promoter for the one or more disclosed isolated nucleic acid molecules can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. In an aspect, a disclosed promoter can be a promoter/enhancer. In an aspect, a disclosed promoter for the one or more disclosed isolated nucleic acid molecules can be an endogenous promoter. In an aspect, a disclosed endogenous promoter can be an endogenous promoter/enhancer. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can generally be obtained from a non-coding region upstream of a transcription initiation site of a gene of interest. In an aspect, a disclosed endogenous promoter or a disclosed endogenous promoter/enhancer can be used for constitutive and efficient expression of a disclosed gene. In an aspect, a disclosed promoter for the one or more disclosed isolated nucleic acid molecules can be a CMV promoter or a CMV promoter/enhancer. CMV promoters and CMV promoters/enhancers are well known to the art. In an aspect, a disclosed promoter can be any eukaryotic RNA polymerase II promoter.

[0205] In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme having IgM specific protease activity. In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can cleave the IgM the heavy chain at the interdomain region betw een constant domains Cp2 and Cp3. In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can cleave human IgM. In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can cleave non-human primate IgM. In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzy me that does not cleave non-human IgM. For example, in an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that does not cleave dog IgM, pig IgM, and mouse IgM. In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed IgM specific recombinant enzyme that does not cleave human IgA, IgD, IgE, IgG, or any combination thereof.

[0206] In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can have a binding domain specific for IgM. In an aspect, a disclosed recombinant enzyme can cleave IgM between position 350 and position 365 of SEQ ID NO: 105. In an aspect, SEQ ID NO: 105 can represent the linear sequence of an IgM (for example, the substrate). In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can have a binding domain capable of specifically binding to one or more amino acid residues 355 through 360 of IgM. In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can have an affinity for human IgM. In an aspect, a disclosed IgM can be cleaved at SEQ ID NO: 110. In an aspect, a disclosed IgM can be cleaved at the sequence comprising 355D. 356T, 357A. 3581. 359R, and 360V. In an aspect, a disclosed IgM can be cleaved at position 355 through position 360 of SEQ ID NO: 105. [0207] In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can have IgG and IgM specific protease activity. In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can cleave the IgM the heavy chain at the interdomain region between constant domains Cp2 and Cp3 and can cleave the IgG below the hinge region. In an aspect, a disclosed recombinant enzyme can cleave the IgG below the hinge region, thereby yielding F(ab’)2 and Fc fragments. In an aspect, a disclosed recombinant enzyme can cleave the IgG separating the Cp2 and Cp3 domains from the Cpl domain.

[0208] In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can cleave human IgG and human IgM. In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can cleave non- human primate IgG and non-human primate IgM. In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme having IgG and IgM protease activity' does not cleave IgA, IgD, IgE, or any combination thereof. In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme having IgG and IgM protease activity does not cleave non-human IgM or non-human IgG. For example, in an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme having IgG and IgM protease activity that does not cleave dog IgM, pig IgM, and mouse IgM, or dog IgG, pig IgG, or mouse IgG.

[0209] In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can have a binding domain capable of specifically binding to the sequence of SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO: 114, or any combination thereof. In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can have a binding domain capable of specifically binding to the sequence of SEQ ID NO: 115. SEQ ID NO: 116, SEQ ID NO: 117. SEQ ID NO: 118, or any combination thereof. In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can cleave IgG at amino acid residues 109 through 124 of the IgG of SEQ ID NO: 106, residues 106 through 120 of the IgG of SEQ ID NO:107, residues 156 through 171 of the IgG of SEQ ID NO: 108, residues 106 through 121 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can cleave IgG at amino acid residues 116 through 122 of the IgG of SEQ ID NO: 106, residues 112 through 118 of the IgG of SEQ ID NO: 107, residues 163 through 169 of the IgG of SEQ ID NO: 108, residues 113 through 119 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, a disclosed vector can comprise the nucleic acid sequence of a disclosed recombinant enzyme that can cleave one or more IgG molecules at SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO:118, or any combination thereof.

[0210] In an aspect, a disclosed encoded recombinant enzyme can clear and/or remove IgM and/or IgM and IgG from circulation in a subject. In an aspect, a disclosed recombinant enzyme can clear and/or remove IgM and/or IgM and IgG from circulation in the sera of a subject. In an aspect, a disclosed recombinant enz me can cleave and/or degrade circulating IgM and/or IgM and IgG in a subject. In an aspect, a disclosed recombinant enzyme can transiently clear and/or remove IgM and/or IgM and IgG from circulation in the sera of a subject, or can transiently cleave and/or degrade circulating IgM and/or IgM and IgG in a subject. In an aspect, a disclosed recombinant enzyme can reduce and/or diminish the level of circulating IgM and/or IgM and IgG levels in a subject, or can transiently reduce and/or diminish the level of circulating IgM and/or IgM and IgG levels in a subject. In aspect, a disclosed transient reduction and/or diminishment can comprise about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks. [0211] In an aspect, a disclosed vector can block and/or minimize activation of the complement cascade. In an aspect, a disclosed vector can block and/or minimize activation of the formation of the C 1 complex and/or C3 complex. In an aspect, a disclosed vector can block and/or minimize activation of the classical complement pathway. In an aspect, a disclosed vector can transiently block and/or minimize activation of the complement cascade, can transiently block and/or minimize activation of the formation of the Cl complex and/or C3 complex, can transiently block and/or minimize activation of the classical complement pathway, or any combination thereof. [0212] In an aspect, a disclosed vector can remove and/or cleave surface-bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells. In an aspect, the disclosed removal and/or cleavage of IgM from the surface of B cells can be reversible and/or transient. In an aspect, a disclosed vector can transiently remove and/or cleave surface-bound IgM from B cells and/or can transiently remove and/or cleave IgM from the surface of B cells. In an aspect, a disclosed vector can remove and/or cleave surface bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells without killing and/or injuring the B cells.

[0213] In an aspect, a disclosed vector can decrease and/or minimize the severity of a rejection event of the one or more transplanted organs. In an aspect a disclosed vector can prevent the rejection of the one or more transplanted organs. In an aspect, a disclosed rejection event can be classified a hyperacute rejection event, an acute rejection event, or a chronic rejection event. In an aspect, following the administration and/or delivery of a disclosed vector, the one or more transplanted organs are not rejected by the subject. In an aspect, following the administration and/or delivery of a disclosed vector, the viability of one or more transplanted organs can be improved and/or increased. In an aspect, following the administration and/or delivery of a disclosed vector, the functionality of one or more transplanted organs can be improved and/or increased. In an aspect, following the administration and/or delivery of a disclosed vector, the functionality⁷ of one or more transplanted organs is not decreased and/or diminished. In an aspect, a disclosed recombinant enzyme can degrade one or more anti-HLA antibodies directed at the one or more transplanted organs.

[0214] In an aspect, a disclosed vector can reduce and/or minimize vector mediated immunotoxicity⁷ in a subject. In an aspect, a disclosed vector can reduce and/or minimize transgene immunogenicity in a subject.

[0215] In an aspect, a disclosed vector can be used to modulate humoral immunity in a subject. In an aspect, modulating humoral immunity in a subject can be an increase in the functionality of the subject’s humoral immunity, or can be a decrease in the functional of the subject’s humoral immunity⁷. [0216] In an aspect, a disclosed vector can enable and/or allow vector re-dosing in a subject. In an aspect, a disclosed aspect, a disclosed vector can improve and/or enhance transgene efficacy and/or expression in a subject. In an aspect, a disclosed vector can improve and/or enhance transgene efficacy and/or expression in a subject. In an aspect, a disclosed vector can improve and/or enhance transgene efficacy and/or expression in a subject. In an aspect, improving and/or enhancing transgene efficacy and/or expression in a subject can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of an improvement and/or enhancement when compared to a pre-existing level of transgene efficacy and/or expression such as, for example, a pre-treatment level (prior to administering one or more disclosed vectors). In an aspect, the amount of improvement and/or enhancement can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as a pre-treatment level (prior to administering one or more disclosed vectors). In an aspect, an improvement and/or enhancement can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having received or not having been administered one or more disclosed vectors). In an aspect, an improvement and/or enhancement can be a partial or incomplete restoration. In an aspect, an improvement and/or enhancement can be complete or near complete such that the level of neutralizing antibodies or neutralizing IgG and/or IgM molecules is similar to that of a wild-type or control level.

4. Pharmaceutical Formulations

[0217] Disclosed herein is a pharmaceutical formulation comprising a disclosed enzyme. Disclosed herein is a pharmaceutical formulation comprising a disclosed enzyme and one or more pharmaceutically acceptable carriers and/or excipients. Disclosed herein is a pharmaceutical formulation comprising a disclosed recombinant enzyme. Disclosed herein is a pharmaceutical formulation comprising a disclosed recombinant enzyme and one or more pharmaceutically acceptable carriers and/or excipients. Disclosed herein is a pharmaceutical formulation comprising a disclosed vector. Disclosed herein is a pharmaceutical formulation comprising a disclosed vector and one or more pharmaceutically acceptable carriers and/or excipients. Disclosed herein is a pharmaceutical formulation comprising an enzyme having IgM specific protease activity. Disclosed herein is a pharmaceutical formulation comprising an enzyme having IgG specific protease activity. Disclosed herein is a pharmaceutical formulation comprising an enzyme having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a recombinant enz me having IgM specific protease activity⁷. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme having IgG specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a recombinant enz me having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a fusion protein having IgM specific protease activity and IgG specific protease activity. Disclosed herein is a pharmaceutical formulation comprising an enzy me comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46. Disclosed herein is a pharmaceutical formulation comprising an enzyme comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46 and having IgM specific protease activity and/or IgG specific protease activity.

[0218] Disclosed herein is a pharmaceutical formulation comprising a disclosed recombinant enzyme having human or non-human primate IgM specific protease activity and a disclosed recombinant enzyme having human or non-human primate IgG specific protease activity. Disclosed herein is a pharmaceutical formulation comprising the recombinant enzyme comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46 and the recombinant enzyme comprising the sequence of SEQ ID NO: 119. Disclosed herein is a pharmaceutical formulation comprising the recombinant enzyme comprising the sequence of any one of SEQ ID NO: 01 - SEQ ID NO:46 and the recombinant enzyme comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein is a pharmaceutical formulation comprising the recombinant enzyme comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46 and the recombinant enzy me comprising the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159. [0219] Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising the sequence of SEQ ID NO: 119. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzy me comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 119. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising the sequence of SEQ ID NO: 119 and having human and non-human primate IgG specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121 and having human and non- human primate IgG specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159 and having human and non-human primate IgG specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 119 and having human and non-human primate IgG specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 120 or SEQ ID NO: 121 and having human and non-human primate IgG specific protease activity. Disclosed herein is a pharmaceutical formulation comprising a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159 and having human and non-human primate IgG specific protease activity.

[0220] Disclosed herein is a pharmaceutical formulation comprising a disclosed isolated nucleic acid molecule. Disclosed herein is a pharmaceutical formulation comprising a disclosed isolated nucleic acid molecule and one or more pharmaceutically acceptable carriers and/or excipients. Disclosed herein is a pharmaceutical formulation comprising an isolated nucleic acid molecule comprising the sequence of any one of SEQ ID NO:47 - SEQ ID NO:92. Disclosed herein is a pharmaceutical formulation comprising an isolated nucleic acid molecule comprising the sequence of any one of SEQ ID NO:47 - SEQ ID NO:92 and one or more pharmaceutically acceptable carriers and/or excipients. Disclosed herein is a pharmaceutical formulation comprising an isolated nucleic acid molecule comprising the sequence of any one of SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a pharmaceutical formulation comprising an isolated nucleic acid molecule comprising the sequence of SEQ ID NO: 160 or SEQ ID NO: 161 and one or more pharmaceutically acceptable carriers and/or excipients.

[0221] In an aspect, a disclosed pharmaceutical formulation can restore one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, a disclosed pharmaceutical formulation can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0222] In an aspect, a disclosed pharmaceutical formulation can have IgM specific protease activity. In an aspect, a disclosed pharmaceutical formulation can cleave the IgM the heavy chain at the interdomain region between constant domains Cp2 and Cp3. In an aspect, a disclosed recombinant enzyme can cleave human IgM. In an aspect, a disclosed pharmaceutical formulation can cleave non-human primate IgM. In an aspect, a disclosed pharmaceutical formulation does not cleave non-human IgM. For example, in an aspect, a disclosed pharmaceutical formulation does not cleave dog IgM, pig IgM, and mouse IgM. In an aspect, a disclosed IgM specific pharmaceutical formulation does not cleave human IgA, IgD, IgE, IgG, or any combination thereof.

[0223] In an aspect, a disclosed pharmaceutical formulation can have a binding domain specific for IgM. In an aspect, a disclosed pharmaceutical formulation can cleave IgM between position 350 and position 365 of SEQ ID NO: 105. In an aspect, SEQ ID NO: 105 can represent the linear sequence of an IgM (for example, the substrate). In an aspect, a disclosed pharmaceutical formulation can have a binding domain capable of specifically binding to one or more amino acid residues 355 through 360 of IgM. In an aspect, a disclosed pharmaceutical formulation can have an affinity for human IgM. In an aspect, a disclosed IgM can be cleaved at SEQ ID NO: 110. In an aspect, a disclosed IgM can be cleaved at the sequence comprising 355D, 356T, 357A, 3581, 359R, and 360V. In an aspect, a disclosed IgM can be cleaved at position 355 through position 360 of SEQ ID NO: 105.

[0224] In an aspect, a disclosed pharmaceutical formulation can have IgG and IgM specific protease activity. In an aspect, a disclosed pharmaceutical formulation can cleave the IgM the heavy chain at the interdomain region between constant domains Cp2 and Cp3 and can cleave the IgG below the hinge region. In an aspect, a disclosed pharmaceutical formulation can cleave the IgG below the hinge region, thereby yielding Ffab'E and Fc fragments. In an aspect, a disclosed pharmaceutical formulation can cleave the IgG separating the Cp2 and Cp3 domains from the Cpl domain.

[0225] In an aspect, a disclosed pharmaceutical formulation can cleave human IgG and human IgM. In an aspect, a disclosed pharmaceutical formulation e can cleave non-human primate IgG and non-human primate IgM. In an aspect, a disclosed pharmaceutical formulation having IgG and IgM protease activity does not cleave IgA, IgD, IgE, or any combination thereof. In an aspect, a disclosed pharmaceutical formulation having IgG and IgM protease activity does not cleave non- human IgM or non-human IgG. For example, in an aspect, a disclosed pharmaceutical formulation having IgG and IgM protease activity does not cleave dog IgM. pig IgM. and mouse IgM, or dog IgG, pig IgG, or mouse IgG.

[0226] In an aspect, a disclosed pharmaceutical formulation can have a binding domain capable of specifically binding to the sequence of SEQ ID NO:111, SEQ ID NO: 112, SEQ ID NO:113, SEQ ID NO: 114. or any combination thereof. In an aspect, disclosed pharmaceutical formulation can have a binding domain capable of specifically binding to the sequence of SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof. In an aspect, disclosed pharmaceutical formulation can cleave IgG at amino acid residues 109 through 124 of the IgG of SEQ ID NO: 106, residues 106 through 120 of the IgG of SEQ ID NO: 107, residues 156 through 171 of the IgG of SEQ ID NO: 108, residues 106 through 121 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, a disclosed pharmaceutical formulation can cleave IgG at amino acid residues 116 through 122 of the IgG of SEQ ID NO:106, residues 112 through 119 of the IgG of SEQ ID NO: 107, residues 163 through 169 of the IgG of SEQ ID NO: 108, residues 113 through 119 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, a disclosed pharmaceutical formulation can cleave one or more IgG molecules at SEQ ID NO:115, SEQ ID NO: 116, SEQ ID NO:117, SEQ ID NO:118, or any combination thereof.

[0227] In an aspect, a disclosed pharmaceutical formulation can clear and/or remove IgM and/or IgG and IgM from circulation in a subject. In an aspect, a disclosed recombinant enzyme can cleave and/or degrade circulating IgM and/or IgG and IgM in a subject. In an aspect, disclosed pharmaceutical formulation can transiently clear and/or remove IgM and/or IgG and IgM from circulation in a subject, or can transiently cleave and/or degrade circulating IgM and/or IgG and IgM in a subject. In an aspect, disclosed pharmaceutical formulation can reduce and/or diminish the level of circulating IgM and/or IgG and IgM levels in a subject, or can transiently reduce and/or diminish the level of circulating IgM and/or IgG and IgM levels in a subject. In aspect, a disclosed transient reduction and/or diminishment can comprise about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[0228] In an aspect, a disclosed pharmaceutical formulation can block and/or minimize activation of the complement cascade. In an aspect, a disclosed pharmaceutical formulation can block and/or minimize activation of the formation of the Cl complex and/or C3 complex. In an aspect, a disclosed pharmaceutical formulation can block and/or minimize activation of the classical complement pathway. In an aspect, a disclosed pharmaceutical formulation can transiently block and/or minimize activation of the complement cascade, can transiently block and/or minimize activation of the formation of the Cl complex and/or C3 complex, can transiently block and/or minimize activation of the classical complement pathway, or any combination thereof.

[0229] In an aspect, a disclosed pharmaceutical formulation can remove and/or cleave surfacebound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells. In an aspect, the disclosed removal and/or cleavage of IgM from the surface of B cells can be reversible and/or transient. In an aspect, a disclosed pharmaceutical formulation can transiently remove and/or cleave surface-bound IgM from B cells and/or can transiently remove and/or cleave IgM from the surface of B cells. In an aspect, a disclosed pharmaceutical formulation me can remove and/or cleave surface bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells without killing and/or injuring the B cells. [0230] In an aspect, a disclosed pharmaceutical formulation can reduce and/or minimize vector mediated immunotoxicity in a subject. In an aspect, a disclosed pharmaceutical formulation can enable and/or allow vector re-dosing in a subject.

[0231] In an aspect, a disclosed pharmaceutical formulation can reduce and/or minimize transgene immunogenicity in a subject. In an aspect, a disclosed pharmaceutical formulation can improve and/or enhance transgene efficacy and/or expression in a subject. In an aspect, improving and/or enhancing transgene efficacy and/or expression in a subject can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of an improvement and/or enhancement when compared to a pre-existing level of transgene efficacy and/or expression such as, for example, a pre-treatment level (prior to administering one or more disclosed pharmaceutical formulations). In an aspect, the amount of improvement and/or enhancement can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as a pre-treatment level (prior to administering one or more disclosed pharmaceutical formulations). In an aspect, an improvement and/or enhancement can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having received or not having been administered one or more disclosed pharmaceutical formulations). In an aspect, an improvement and/or enhancement can be a partial or incomplete restoration. In an aspect, an improvement and/or enhancement can be complete or near complete such that the level of neutralizing antibodies or neutralizing IgG and/or IgM molecules is similar to that of a wild-type or control level.

[0232] In an aspect, a disclosed pharmaceutical formulation can comprise (i) one or more active agents, (ii) biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed composition can comprise one or more immune modulators. In an aspect, a disclosed composition can comprise one or more proteasome inhibitors. In an aspect, a disclosed composition can comprise one or more immunosuppressives or immunosuppressive agents. In an aspect, an immunosuppressive agent can be anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), or a combination thereof. In an aspect, a disclosed formulation can comprise an anaplerotic agent (such as, for example, C7 compounds like triheptanoin or MCT).

[0233] In an aspect, a pharmaceutical formulation can decrease and/or minimize the severity of a rejection event of the one or more transplanted organs. In an aspect, a pharmaceutical formulation can prevent the rejection of the one or more transplanted organs. In an aspect, a disclosed rejection event can be classified a hyperacute rejection event, an acute rejection event, or a chronic rejection event. In an aspect, following administration of a disclosed pharmaceutical formulation, a disclosed isolated nucleic acid molecule, the one or more transplanted organs are not rejected by the subject. In an aspect, following administration of a disclosed pharmaceutical formulation, the viability of one or more transplanted organs can be improved and/or increased. In an aspect, following administration of a disclosed pharmaceutical formulation, the functionality of one or more transplanted organs can be improved and/or increased. In an aspect, following administration of a disclosed pharmaceutical formulation, the functionality of one or more transplanted organs is not decreased and/or diminished. In an aspect, a disclosed pharmaceutical formulation can degrade one or more anti-HLA antibodies directed at the one or more transplanted organs. In an aspect, any disclosed pharmaceutical formulation can comprise one or more excipients and/or pharmaceutically acceptable carriers. Excipients and/or pharmaceutically acceptable carriers are known to the art and are discussed supra.

[0234] In an aspect of a disclosed pharmaceutical formulation, a disclosed enzyme or disclosed recombinant enzyme can be therapeutically effective when the dose comprises about 0.01 mg/kg body weight to about 100 mg/kg body weight. In an aspect, a disclosed enzyme or disclosed recombinant enzyme can be therapeutically effective when the dose comprises about 0.01 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg body weight.

5. Plasmids

[0235] Disclosed herein is a plasmid comprising one or more disclosed isolated nucleic acid molecules. Disclosed herein is a plasmid comprising one or more disclosed vectors. Disclosed here are plasmids used in methods of making a disclosed composition such as, for example, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. Plasmids and using plasmids are known to the art.

[0236] Disclosed herein is a plasmid comprising the sequence set forth in any one of SEQ ID NO:93 or a fragment thereof. Disclosed herein is a plasmid comprising a sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in SEQ ID NO:93 or a fragment thereof. Disclosed herein is a plasmid comprising a sequence having at least 40%-60%, at least 60%-80%, at least 80%-90%, or at least 90%-100% identity to the sequence set forth in SEQ ID NO:93 or a fragment thereof.

[0237] Disclosed herein is a plasmid comprising a nucleic acid sequence set forth in any one of SEQ ID NO:47 - SEQ ID NO: 92. Disclosed herein is a plasmid comprising a nucleic acid molecule that, upon expression, can generate an enzyme comprising the sequence of any one of SEQ ID NO: 01 - SEQ ID NO: 46. Disclosed herein is a plasmid comprising a nucleic acid sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a plasmid comprising a nucleic acid molecule that, upon expression, can generate an enzyme comprising the sequence of any one of SEQ ID NO: 119 - SEQ ID NO: 121.

6. Cells

[0238] Disclosed herein are cells comprising a disclosed isolated nucleic acid molecule, a disclosed vector, and/or a disclosed plasmid. Disclosed herein are cells transduced by one or more disclosed viral vectors. Disclosed herein are cells transduced by one or more disclosed recombinant viral vectors. Disclosed herein are cells transfected with one or more disclosed isolated nucleic acid molecules. In an aspect, a disclosed cell has been transfected or disclosed cells have been transfected with one or more nucleic acid sequences having the sequence set forth in SEQ ID NO 93.

[0239] Disclosed herein are cells that have been transfected by a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO:47 - SEQ ID NO: 92. Disclosed herein are cells that generate a disclosed enzyme or a disclosed recombinant enzyme for harvesting and purification. Disclosed herein are cells that generate an enzyme comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46 for harvesting and purification. Disclosed herein are cells that, upon expression of the nucleic acid molecule comprising the sequence of any one of SEQ ID NO:47 - SEQ ID NO:92, can generate an enzyme comprising the sequence of any one of SEQ ID NO: 01 - SEQ ID NO:46 for harvesting and purification.

[0240] Disclosed herein are cells that have been transfected by a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein are cells that generate a disclosed enzyme or a disclosed recombinant enz me for harvesting and purification. Disclosed herein are cells that generate an enzy me comprising the sequence of any one of SEQ ID NO: 119 - SEQ ID NO: 121 for harvesting and purification. Disclosed herein are cells that generate an enzyme comprising the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159 for harvesting and purification. Disclosed herein are cells that, upon expression of the nucleic acid molecule comprising the sequence of SEQ ID NO:160 or SEQ ID NO: 161, can generate an enzyme comprising the sequence of SEQ ID NO: 119 for harvesting and purification.

[0241] In an aspect, a disclosed cell or disclosed cells can be humanized. Techniques to achieve transfection and transduction are known to the art and using transfected or transduced cells are known to the art. In an aspect, disclosed herein are human immortalized cells lines transduced by one or more disclosed viral vectors or transfected with one or more disclosed isolated nucleic acids or disclosed plasmids. In an aspect, disclosed herein are human immortalized cells lines contacted with one or more disclosed pharmaceutical formulations. Disclosed herein are cells obtained for a subject treated with one or more disclosed isolated nucleic acid molecule, one or more disclosed vectors, one or more disclosed plasmids, or one or more disclosed pharmaceutical formulations.

7. Animals

[0242] Disclosed herein are animals treated with one or more disclosed enzymes, one or more disclosed recombinant enzymes, one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, and/or one or more disclosed plasmids. Transgenic animals are known to the art as are the techniques to generate transgenic animals.

C. Methods of Cleaving IgM and/or IgG

[0243] Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with one or more disclosed enzymes or disclosed recombinant enzymes. Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with one or more disclosed nucleic acid molecules. Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with one or more disclosed vectors. Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with one or more disclosed pharmaceutical formulations. [0244] Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with an enzyme comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO: 15. Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with an enzyme comprising the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:38. Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with an enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03. Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with an enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06. Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with an enzyme comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09. Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with an enzy me comprising the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with an enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15.

[0245] Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with one or more disclosed nucleic acid molecules comprising the sequence set forth in any one of SEQ ID NO:47 - SEQ ID NO:61. Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with one or more disclosed nucleic acid molecules comprising the sequence set forth in any one of SEQ ID NO:70 - SEQ ID NO:84. Disclosed herein is a method of cleaving immunoglobulin M (IgM). the method comprising contacting one or more IgM molecules with one or more disclosed nucleic acid molecules comprising the sequence set forth in SEQ ID NO:48 or SEQ ID NO:49. Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with one or more disclosed nucleic acid molecules comprising the sequence set forth in SEQ ID NO:51 or SEQ ID NO:52. Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with one or more disclosed nucleic acid comprising the sequence set forth in SEQ ID NO:54 or SEQ ID NO:55. Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with one or more disclosed nucleic acid comprising the sequence set forth in SEQ ID NO:57 or SEQ ID NO:58. Disclosed herein is a method of cleaving immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules with one or more disclosed nucleic acid comprising the sequence set forth in SEQ ID NO: 60 or SEQ ID NO:61.

[0246] Disclosed herein is a method of cleaving immunoglobulin G (IgG), the method comprising contacting one or more IgG molecules with one or more disclosed nucleic acid molecules comprising the sequence set forth in SEQ ID NO: 160 - SEQ ID NO: 161. Disclosed herein is a method of cleaving immunoglobulin G (IgG), the method comprising contacting one or more IgG molecules with one or more disclosed nucleic acid molecules comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a method of cleaving immunoglobulin G (IgG), the method comprising contacting one or more IgG molecules with one or more disclosed nucleic acid molecules comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a method of cleaving immunoglobulin G (IgG), the method comprising contacting one or more IgG molecules with one or more disclosed nucleic acid molecules comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a method of cleaving immunoglobulin G (IgG), the method comprising contacting one or more IgG molecules with one or more disclosed nucleic acid comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a method of cleaving immunoglobulin G (IgG), the method comprising contacting one or more IgG molecules with one or more disclosed nucleic acid comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. Disclosed herein is a method of cleaving immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules with one or more disclosed nucleic acid comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161.

[0247] Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with one or more disclosed enzymes or disclosed recombinant enzy mes. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with one or more disclosed nucleic acid molecules. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with one or more disclosed vectors. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with one or more disclosed pharmaceutical formulations. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with an enzy me comprising the sequence set forth in any one of SEQ ID NO: 16 - SEQ ID NO:23. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with an enzy me comprising the sequence set forth in any one of SEQ ID NO: 39 - SEQ ID NO:46. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with an enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with an enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with an enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with an enzyme comprising the sequence set forth in any one of SEQ ID NO:39 - SEQ ID NO:46. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with an enzyme comprising the sequence set forth in any one of SEQ ID NO: 119 - SEQ ID NO: 121.

[0248] Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO:62 - SEQ ID NO:69. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO: 85 - SEQ ID NO:92. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:63 or SEQ ID NO:64. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:66 or SEQ ID NO:67. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:68 or SEQ ID NO:69. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 86 or SEQ ID NO: 87. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 89 or SEQ ID NQ:90. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:91 or SEQ ID NO:92.

[0249] In an aspect, the IgM molecule and/or the IgG molecule that is cleaved can be in the sera of a subject. In an aspect, the IgM molecule and/or the IgG molecule that is cleaved can be circulating in a subject. In an aspect, a disclosed IgM molecule and/or the IgG molecule can be neutralizing against an exogenously administered protein. In an aspect, a disclosed IgM molecule and/or the IgG molecule can be neutralizing against enzyme replacement therapy or protein replacement therapy or recombinant product. In an aspect, a disclosed IgM molecule and/or the IgG molecule can be neutralizing against a non-viral vector or a viral vector. In an aspect, a disclosed IgM molecule and/or the IgG molecule can be neutralizing against a protein associated with a non-viral vector or a viral vector. For example, in an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against a viral vector capsid protein (e.g., an AAV capsid protein).

[0250] In an aspect, a disclosed method can further comprise diagnosing a subject as being in need of one or more disclosed enzymes, one or more disclosed recombinant enzymes, one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0251] In an aspect of a disclosed method, there is at least a 10% reduction, at least a 20% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, at least an 80% reduction, at least a 90% reduction, or at least a 99% reduction in the level of circulating intact IgM and/or intact IgG.

[0252] In an aspect, a disclosed IgM molecule and/or the IgG molecule can be neutralizing against one or more transplanted organs. In an aspect, a disclosed method can decrease and/or can minimize the severity’ of a rejection event of the one or more transplanted organs. In an aspect, a disclosed method can prevent the rejection of the one or more transplanted organs. In an aspect, a disclosed rejection event can be classified ahyperacute rejection event, an acute rejection event, or a chronic rejection event. In an aspect, a disclosed method can reduce the risk of rejection of the one or more transplanted organs. In an aspect, a disclosed method can reduce or diminish the subject's need for long-term immunosuppression. In an aspect, a disclosed method can improve and/or increase the viability of one or more transplanted organs. In an aspect, a disclosed method can improve and/or increase the functionality of one or more transplanted organs. In an aspect, a disclosed method does not decrease and/or diminish the functionality of one or more transplanted organs. In an aspect, a disclosed method can degrade one or more anti-HLA antibodies directed at the one or more transplanted organs.

[0253] In an aspect, a subject can have a high sustained antibody titer (HSAT - defined as titers of > 12,800). In an aspect, a subject can have a high sustained antibody titer (HSAT) against one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more non-viral vectors or viral vectors, one or more proteins associated with a non-viral vector, one or more proteins associated with a viral vector, or any combination thereof. In an aspect, after the contacting step, a disclosed HSAT can be reduced and/or eliminated. In an aspect, after the reduction and/or elimination of one or more HSATs, a subject can be re-dosed. In an aspect, after the reduction and/or elimination of one or more HSATs, a subject can be re-dosed with one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more recombinant products, one or more non-viral vectors or viral vectors, one or more proteins associated with a non-viral vector, one or more proteins associated with a viral vector, or any combination thereof.

[0254] In an aspect, a disclosed method can comprise modifying one or more of the disclosed steps. For example, modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subj ect. or by changing the frequency of administration of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject, or by changing the duration of time one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered to a subject.

[0255] In an aspect, a disclosed method can further comprise measuring one or more time the levels of IgM and/or IgG in the subject (such as, for example, in the subject’s sera).

[0256] In an aspect, a disclosed method of cleaving IgM and/or IgG can clear and/or remove IgM and/or IgG from circulation in a subject. In an aspect, a disclosed method of cleaving IgM and/or IgG can leave and/or degrade circulating IgM and/or IgG in a subject. In an aspect, a disclosed method can transiently clear and/or remove IgM and/or IgG from circulation in a subject, or can transiently cleave and/or degrade circulating IgM and/or IgG in a subject. In an aspect, a disclosed method of cleaving IgM and/or IgG can reduce and/or diminish the level of circulating IgM and/or IgG levels in a subject, or can transiently reduce and/or diminish the level of circulating IgM and/or IgG levels in a subject. In aspect, a disclosed transient reduction and/or diminishment can comprise about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[0257] In an aspect, a disclosed method of cleaving IgM and/or IgG can block and/or minimize activation of the complement cascade. In an aspect, a disclosed method of cleaving IgM and/or IgG can block and/or minimize activation of the formation of the Cl complex and/or C3 complex. In an aspect, a disclosed method of cleaving IgM and/or IgG can block and/or minimize activation of the classical complement pathway. In an aspect, a disclosed method can transiently block and/or minimize activation of the complement cascade, can transiently block and/or minimize activation of the formation of the Cl complex and/or C3 complex, can transiently block and/or minimize activation of the classical complement pathway, or any combination thereof.

[0258] In an aspect, a disclosed method of cleaving IgM and/or IgG can remove and/or cleave surface-bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells. [0259] In an aspect, the disclosed removal and/or cleavage of IgM from the surface of B cells can be reversible and/or transient. In an aspect, a disclosed method of cleaving IgM and/or IgG can transiently remove and/or cleave surface-bound IgM from B cells and/or can transiently remove and/or cleave IgM from the surface of B cells. In an aspect, a disclosed method can remove and/or cleave surface bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells without killing and/or injuring the B cells.

[0260] In an aspect, a disclosed method of cleaving IgM and/or IgG can reduce and/or minimize vector mediated immunotoxicity in a subject. In an aspect, a disclosed method of cleaving IgM and/or IgG can enable and/or allow vector re-dosing in a subject.

[0261] In an aspect, a disclosed method of cleaving IgM and/or IgG can modulate humoral immunity in a subject. In an aspect, modulating humoral immunity in a subject can be an increase in the functionality of the subject’s humoral immunity, or can be a decrease in the functional of the subject's humoral immunity.

[0262] In an aspect of a disclosed method, intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an administered gene therapy. In an aspect of a disclosed method, intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an administered therapeutic protein or a therapeutic recombinant protein. In an aspect of a disclosed method, the intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an encoded therapeutic protein or encoded therapeutic recombinant protein.

[0263] In an aspect, a disclosed method of cleaving IgM and/or IgG can reduce and/or minimize transgene immunogenicity in a subject. In an aspect, a disclosed method of cleaving IgM and/or IgG can improve and/or enhance transgene efficacy and/or expression in a subject. In an aspect, improving and/or enhancing transgene efficacy and/or expression in a subject can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of an improvement and/or enhancement when compared to a pre-existing level of transgene efficacy and/or expression such as, for example, a pre-treatment level (prior to administering one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, the amount of improvement and/or enhancement can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%. 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as a pre-treatment level (prior to administering one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, an improvement and/or enhancement can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having received or not having been administered one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, an improvement and/or enhancement can be a partial or incomplete restoration. In an aspect, an improvement and/or enhancement can be complete or near complete such that the level of neutralizing antibodies or neutralizing IgG and/or IgM molecules is similar to that of a wildtype or control level.

[0264] In an aspect of a disclosed method, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered prior to, concurrent, or after the delivery and/or administration of enz me replacement therapy, protein replacement, gene therapy, a recombinant product, or any combination thereof.

[0265] In an aspect, a disclosed method of cleaving IgM and/or IgG can further comprise reducing and/or minimizing vector-mediated immunotoxicity and/or transgene immunogenicity (e.g., the ability to induce specific immunity). In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the efficacy of the recombinant product encoded by the transgene. In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subject with one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subject with gene therapy, enzyme replacement therapy, protein replacement, recombinant product, or any combination thereof.

[0266] In an aspect of a disclosed method of cleaving IgM and/or IgG, IgM can be cleaved in the heavy chain at the interdomain region between constant domains Cp2 and Cp3. In an aspect, IgM can be cleaved between position 350 and position 365 of SEQ ID NO: 105. In an aspect, SEQ ID NO: 105 can represent the linear sequence of an IgM (for example, the substrate). In an aspect, IgM can be cleaved between positions 355 through 360 of IgM. In an aspect, IgM can be cleaved at SEQ ID NO: 110. In an aspect, IgM can be cleaved at the sequence comprising 355D, 356T, 357 A, 3581, 359R, and 360V. In an aspect IgM can be cleaved at position 355 through position 360 of the IgM of SEQ ID NO: 105. In an aspect, IgG can be cleaved below the hinge region, thereby yielding F(ab’)2 and Fc fragments. In an aspect, IgG can be cleaved to separate the Cu2 and Cp3 domains from the Cpl domain. In an aspect, IgG can be cleaved at SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 1 13, SEQ ID NO: 114. or any combination thereof. In an aspect, IgG can be cleaved at SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 1 17, SEQ ID NO: 118, or any combination thereof. In an aspect, IgG can be cleaved at positions 109 through 124 of the IgG of SEQ ID NO: 106, positions 106 through 120 of the IgG of SEQ ID NO: 107, positions 156 through 171 ofthe lgG of SEQ ID NO: 108, positions 106 through 121 of lgG of SEQ ID NO: 109, or any combination thereof. In an aspect, IgG can be cleaved at positions 116 through 122 of the IgG of SEQ ID NO: 106, positions 112 through 118 of the IgG of SEQ ID NO: 107, positions 163 through 169 of the IgG of SEQ ID NO: 108, positions 113 through 119 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, IgG can be cleaved SEQ ID NO: 115. SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof.

[0267] Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with one or more disclosed recombinant enzymes having IgM specific protease activity and one or more disclosed recombinant enzymes having IgG specific protease activity.

[0268] Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with one or more disclosed recombinant enzymes comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46 and one or more disclosed recombinant enzymes comprising the sequence of any one SEQ ID NO: 119 - SEQ ID NO: 159.

[0269] Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 11 or SEQ ID NO: 12 and one or more disclosed recombinant enzymes comprising the sequence of any one SEQ ID NO: 119 - SEQ ID NO: 159. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 11 or SEQ ID NO: 12 and one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 1 19. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 14 or SEQ ID NO: 15 and one or more disclosed recombinant enzymes comprising the sequence of any one SEQ ID NO: 119 - SEQ ID NO: 159. Disclosed herein is a method of cleaving immunoglobulin M (IgM) and immunoglobulin G (IgG), the method comprising contacting one or more IgM molecules and IgG molecules with one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 14 or SEQ ID NO: 15 and one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO:119.

[0270] In an aspect, a disclosed method of cleaving IgM and/or IgG can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of SEQ ID NO: 119. In an aspect, a disclosed method of cleaving IgM and/or IgG can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. In an aspect, a disclosed method of cleaving IgM and/or IgG can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159. In an aspect, a disclosed method of cleaving IgM and/or IgG can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 119. In an aspect, a disclosed method of cleaving IgM and/or IgG can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. In an aspect, a disclosed method of cleaving IgM and/or IgG can further comprise contacting one or more IgM molecules and IgG molecules with a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159. [0271] In an aspect, a disclosed method can further comprise administering one or more times one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0272] In an aspect, a disclosed method can further comprise measuring and/or determining a subject’s pre-treatment level of circulating IgG and/or IgM and/or a subject’s pre-treatment level of surface bound IgM. In an aspect, a disclosed method can further comprise measuring and/or determining one or more times a subject’s level of circulating IgG and/or IgM and/or a subject’s level of surface bound IgM (e.g., before, during, and after a disclosed administering step).

[0273] In an aspect, a disclosed method can be performed using a singular disclosed enzyme having both IgG and IgM specific protease activity. In an aspect, a disclosed method can be performed using a disclosed enzyme having IgG specific protease activity and a disclosed enzyme having IgM specific protease activity.

D. Methods of Cleaving Surface Bound IgM

[0274] Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed enzymes or disclosed recombinant enzymes. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed nucleic acid molecules. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed vectors. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed pharmaceutical formulations. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with an enzyme comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO: 15. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with an enzyme comprising the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:38. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with an enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with an enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with an enzyme comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with an enzyme comprising the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with an enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15.

[0275] Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed nucleic acid molecules comprising the sequence set forth in any one of SEQ ID NO:47 - SEQ ID N0:61. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed nucleic acid molecules comprising the sequence set forth in any one of SEQ ID NO:70 - SEQ ID NO: 84.

[0276] Disclosed herein is a method of cleaving bound immunoglobulin M (IgM). the method comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed nucleic acid molecules comprising the sequence set forth in SEQ ID NO:48 or SEQ ID NO:49. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed nucleic acid molecules comprising the sequence set forth in SEQ ID NO:51 or SEQ ID NO:52. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed nucleic acid comprising the sequence set forth in SEQ ID NO:54 or SEQ ID NO:55. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed nucleic acid comprising the sequence set forth in SEQ ID NO:57 or SEQ ID NO:58. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with one or more disclosed nucleic acid comprising the sequence set forth in SEQ ID NO:60 or SEQ ID NO:61.

[0277] Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with an enzyme comprising the sequence set forth in any one of SEQ ID NO: 16 - SEQ ID NO:23. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with an enzyme comprising the sequence set forth in any one of SEQ ID NO:39 - SEQ ID NO:46. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with an enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO:18. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with an enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with an enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM). the method comprising contacting one or more IgM molecules bound to the surface of B cells with an enzyme comprising the sequence set forth in any one of SEQ ID NO:39 - SEQ ID NO:46.

[0278] Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO:62- SEQ ID NO:69. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO:85 - SEQ ID NO:92. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM). the method comprising contacting one or more IgM molecules bound to the surface of B cells with anucleic acid molecule comprising the sequence set forth in SEQ ID NO:63 or SEQ ID NO:64. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:66 or SEQ ID NO:67. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells w ith a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:68 or SEQ ID NO:69. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM). the method comprising contacting one or more IgM molecules bound to the surface of B cells with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:86 or SEQ ID NO:87. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 89 or SEQ ID NQ:90. Disclosed herein is a method of cleaving bound immunoglobulin M (IgM), the method comprising contacting one or more IgM molecules bound to the surface of B cells with a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:91 or SEQ ID NO:92.

[0279] In an aspect, disclosed B cells can be in the sera of a subject. In an aspect, disclosed B cells can be circulating in a subject.

[0280] In an aspect, a disclosed surface-bound IgM molecule can be neutralizing against an exogenously administered protein. In an aspect, a disclosed surface-bound IgM molecule can be neutralizing against enzy me replacement therapy or protein replacement therapy or recombinant product. In an aspect, a disclosed surface-bound IgM molecule can be neutralizing against a non- viral vector or a viral vector. In an aspect, a disclosed surface-bound IgM molecule can be neutralizing against a protein associated with a non-viral vector or a viral vector. For example, in an aspect, a disclosed surface-bound IgM molecule can be neutralizing against a viral vector capsid protein (e.g., an AAV capsid protein). [0281] In an aspect, a disclosed method can further comprise diagnosing a subject as being in need of one or more disclosed enzymes, one or more disclosed recombinant enzymes, one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0282] In an aspect of a disclosed method, there is at least a 10% reduction, at least a 20% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, at least an 80% reduction, at least a 90% reduction, or at least a 99% reduction in the level of circulating intact IgM and/or intact IgG.

[0283] In an aspect, a disclosed surface-bound IgM molecule can be neutralizing against one or more transplanted organs. In an aspect, a disclosed method can degrade one or more anti-HLA antibodies directed at the one or more transplanted organs.

[0284] In an aspect, a subject can have a high sustained antibody titer (HSAT - defined as titers of > 12,800). In an aspect, a subject can have a high sustained antibody titer (HSAT) against one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more non-viral vectors or viral vectors, one or more proteins associated with a non-viral vector, one or more proteins associated with a viral vector, or any combination thereof. In an aspect, after the contacting step, a disclosed HSAT can be reduced and/or eliminated. In an aspect, after the reduction and/or elimination of one or more HS ATs, a subject can be re-dosed. In an aspect, after the reduction and/or elimination of one or more HSATs, a subject can be re-dosed with one or more exogenously administered proteins or enzy mes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more recombinant products, one or more non-viral vectors or viral vectors, one or more proteins associated with a non-viral vector, one or more proteins associated with a viral vector, or any combination thereof.

[0285] In an aspect, a disclosed method can further comprise measuring one or more time the levels of IgM and/or IgG in the subject (such as, for example, in the subject’s sera).

[0286] In an aspect, a disclosed method of cleaving surface-bound IgM can clear and/or remove IgM from circulation in a subject. In an aspect, a disclosed method of cleaving surface-bound IgM can cleave and/or degrade circulating IgM in a subject. In an aspect, a disclosed method of cleaving surface-bound IgM can transiently clear and/or remove IgM from circulation in a subject, or can transiently cleave and/or degrade circulating IgM in a subject. In an aspect, a disclosed method of cleaving surface-bound IgM can reduce and/or diminish the level of circulating IgM levels in a subj ect, or can transiently⁷ reduce and/or diminish the level of circulating IgM levels in a subject. In an aspect, a disclosed method can remove and/or cleave surface bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells without killing and/or injuring the B cells.

[0287] In aspect, a disclosed transient reduction and/or diminishment can comprise about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[0288] In an aspect, a disclosed method of cleaving surface-bound IgM can block and/or minimize activation of the complement cascade. In an aspect, a disclosed method of cleaving surface-bound IgM can block and/or minimize activation of the formation of the C 1 complex and/or C3 complex. In an aspect, a disclosed method of cleaving surface-bound IgM can block and/or minimize activation of the classical complement pathway.

[0289] In an aspect, a disclosed method of cleaving surface-bound IgM can transiently block and/or minimize activation of the complement cascade, can transiently block and/or minimize activation of the formation of the Cl complex and/or C3 complex, can transiently block and/or minimize activation of the classical complement pathway, or any combination thereof.

[0290] In an aspect, a disclosed method of cleaving surface-bound IgM can reduce and/or minimize vector mediated immunotoxicity in a subject. In an aspect, a disclosed method of cleaving surface-bound IgM can enable and/or allow vector re-dosing in a subject. In an aspect, a disclosed method of cleaving surface-bound IgM can enable and/or allow re-dosing of one or more disclosed enzymes or recombinant enzy mes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof in a subject.

[0291] In an aspect, a disclosed method of cleaving surface-bound IgM can reduce and/or minimize transgene immunogenicity in a subject. In an aspect, a disclosed method of cleaving surface-bound IgM can improve and/or enhance transgene efficacy and/or expression in a subject. In an aspect, improving and/or enhancing transgene efficacy and/or expression in a subject can comprise a 10%, 20%, 30%, 40%, 50%. 60%, 70%, 80%, 90%, 100%, or any amount of an improvement and/or enhancement when compared to a pre-existing level of transgene efficacy and/or expression such as, for example, a pre-treatment level (prior to administering one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, the amount of improvement and/or enhancement can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as a pre-treatment level (prior to administering one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, an improvement and/or enhancement can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having received or not having been administered one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, an improvement and/or enhancement can be a partial or incomplete restoration. In an aspect, an improvement and/or enhancement can be complete or near complete such that the level of neutralizing antibodies or neutralizing IgG and/or IgM molecules is similar to that of a wildtype or control level.

[0292] In an aspect, a disclosed method of cleaving surface-bound IgM can modulate humoral immunity in a subj ect. In an aspect, modulating humoral immunity' in a subject can be an increase in the functionality of the subject's humoral immunity, or can be a decrease in the functional of the subject’s humoral immunity.

[0293] In an aspect of a disclosed method, intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an administered gene therapy. In an aspect of a disclosed method, intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an administered therapeutic protein or a therapeutic recombinant protein. In an aspect of a disclosed method, the intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an encoded therapeutic protein or encoded therapeutic recombinant protein.

[0294] In an aspect, a disclosed method can comprise administering to a subject in need thereof a theoretically effectively amount of one or more disclosed enzymes or disclosed recombinant enzymes, one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0295] In an aspect, a therapeutically effective amount of a disclosed enzyme or disclosed recombinant enzyme can comprise about 0.01 mg/kg body weight to about 100 mg/kg body weight. In an aspect, a disclosed enzyme or disclosed recombinant enzyme can be therapeutically effective when the dose comprises about 0.01 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg body weight. [0296] In an aspect a subject can have or can be suspected of having one of more disclosed autoimmune diseases and/or disorders, one or more autoinflammatory diseases and/or disorders, one or more inflammatory diseases and/or disorders, or any combination thereof.

[0297] In an aspect, a disclosed autoimmune disease and/or disorder can be celiac disease, diabetes mellitus type 1. Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, or any combination thereof.

[0298] In an aspect, a disclosed autoinflammatory diseases and/or disorder can be Familial Mediterranean Fever (FMF), hyper IgD syndrome (HIDS), TNF receptor-associated periodic syndrome (TRAPS), cryopyrin associated periodic syndromes (CAPS), Blau syndrome, pyogenic sterile arthritis pyoderma gangrenosum and acne syndrome (PAPA), or any combination thereof. [0299] In an aspect, a disclosed inflammatory disease and/or disorder can be Encephalitis, Myelitis, Meningitis, Arachnoiditis, Neuritis, Dacryoadenitis, Scleritis, Episcleritis, Keratitis, Retinitis, Chorioretinitis, Blepharitis, Conjunctivitis, Uveitis, Otitis externa, Otitis media, Labyrinthitis, Mastoiditis, Carditis, Endocarditis, Myocarditis, Pericarditis, Vasculitis, Arteritis, Phlebitis, Capillaritis, Sinusitis, Rhinitis, Pharyngitis, Laryngitis Tracheitis, Bronchitis, Bronchiolitis, Pneumonitis, Pleuritis, Mediastinitis, Stomatitis, Gingivitis, Gingivostomatitis, Glossitis, Tonsillitis, Sialadenitis/Parotitis, Cheilitis, Pulpitis, Gnathitis, Esophagitis, Gastritis, Gastroenteritis. Enteritis, Colitis, Enterocolitis, Duodenitis. Ileitis. Caecitis, Appendicitis, Proctitis, Hepatitis, Ascending cholangitis, Cholecystitis, Pancreatitis, Peritonitis, Dermatitis, Folliculitis, Cellulitis, Hidradenitis, Arthritis, Dermatomyositis, Myositis, Synovitis/Tenosynovitis, Bursitis, Enthesitis, Fasciitis, Capsulitis, Epicondylitis, Tendinitis, Panniculitis, Osteochondritis, Spondylitis. Periostitis, Chondritis. Nephritis, Glomerulonephritis, Pyelonephritis, Ureteritis, Cystitis, Urethritis, Oophoritis, Salpingitis, Endometritis, Parametritis, Cervicitis, Vaginitis, Vulvitis, Mastitis, Orchitis, Epididymitis, Prostatitis, Seminal vesiculitis. Balanitis, Posthitis, Balanoposthitis, Chorioamnionitis, Funisitis, Omphalitis, Insulitis, Hypophysitis, Thyroiditis. Parathyroiditis, Adrenalitis, Lymphangitis, Lymphadenitis, or any combination thereof.

[0300] In an aspect, a subject can be in need of one or more organ transplants. In an aspect, a subj ect can have received one or more organ transplants. In an aspect, a disclosed organ transplant can comprise the heart, one or both kidneys, the liver, one or both lungs, the pancreas, part or all of the colon or the intestines, or any combination thereof.

[0301] In an aspect, a subject can be in need of gene therapy, can have received gene therapy, or can be receiving gene therapy. In an aspect, a subj ect can be treated with gene therapy or can have a disease and/or disorder that can be treated with gene therapy. [0302] In an aspect, a disclosed gene therapy can comprise the delivery and/or repair of one or more disclosed genes. In an aspect, a disclosed gene therapy can comprise AAV-based gene therapy, Adenovirus-based gene therapy, Lentiviral gene therapy, or the combination thereof.

[0303] In an aspect, examples of such genetic diseases and/or disorders or diseases and/or disorders that can be treated with gene therapy include those discussed supra at Part VII(B)(2).

[0304] In an aspect, a disclosed gene therapy can comprise the delivery and/or repair of one or more disclosed genes. Genetic diseases and/or disorders can be due to a defect in one or more genes discussed supra at Part VII(B)(2).

[0305] In an aspect, a disclosed gene therapy can comprise the delivery and/or repair of one or more disclosed genes. Genetic diseases and/or disorders can be due to a defect in one or more genes discussed supra at Part VI(B)(2).

[0306] In an aspect, a disclosed method of cleaving surface-bound IgM can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme delivered by gene therapy. In an aspect, a disclosed method of cleaving surface-bound IgM can comprise restoring one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation.

[0307] In an aspect, restoring one or more aspect of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation comprises restoring the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0308] In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy’ pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.

[0309] In an aspect, restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.

[0310] In an aspect, a disclosed method of cleaving surface-bound IgM, techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are provided below.

[0311] In an aspect of a disclosed method of cleaving surface-bound IgM, contacting a cell can comprising methods known to the art. For example, contacting can comprise administering to a subject one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof .

[0312] In an aspect, administering can comprise intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intra-CSF, intrathecal, intraventricular, intrahepatic. hepatic intraarterial, hepatic portal vein (HPV), or in utero administration. In an aspect, a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be administered via intra-CSF administration in combination with RNAi, antisense oligonucleotides, miRNA, one or more small molecules, one or more therapeutic agents, one or more proteasome inhibitors, one or more immune modulators, and/or a gene editing system. In an aspect, a disclosed composition, a disclosed enzy me or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be administered via LNP administration. In an aspect, a disclosed composition, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be concurrently and/or serially administered to a subject via multiple routes of administration. For example, in an aspect, administering a disclosed nucleic acid molecule, a disclosed enzyme or disclosed recombinant enzyme, a disclosed vector, and/or a disclosed pharmaceutical formulation can comprise intravenous administration and intra-cistem magna (ICM) administration. In an aspect, administering a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can comprise IV administration and intrathecal (ITH) administration. In an aspect, a disclosed method can employ multiple routes of administration to the subject. In an aspect, a disclosed method can employ a first route of administration that can be the same or different as a second and/or subsequent routes of administration.

[0313] In an aspect, a therapeutically effective amount of a disclosed enzyme or disclosed recombinant enzyme can comprise about 0.01 mg/kg body weight to about 100 mg/kg body weight. In an aspect, a disclosed enzyme or disclosed recombinant enzy me can be therapeutically effective when the dose comprises about 0.01 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg body weight.

[0314] In an aspect of a disclosed method of cleaving surface-bound IgM, a therapeutically effective amount of disclosed vector can be delivered to a subject via intravenous (IV) administration and can comprise a range of about 1 x 10¹⁰ vg/kg to about 2 x 10¹⁴ vg/kg. In an aspect, for example, a disclosed vector can be administered at a dose of about 1 x 10¹¹ vg/kg to about 8 x 10° vg/kg or about 1 x 10¹² vg/kg to about 8 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of about I x 10¹³ vg/kg to about 6 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of at least about 1 x 10¹⁰ vg/kg, at least about 5 x IO¹⁰ vg/kg, at least about 1 x 10¹¹ vg/kg, at least about 5 x I0¹¹ vg/kg, at least about I x IO¹² vg/kg, at least about 5 x 10¹² vg/kg, at least about I x 10¹³ vg/kg, at least about 5 x 10¹³ vg/kg, or at least about I x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered at a dose of no more than about 1 x 10¹⁰ vg/kg, no more than about 5 x 10¹⁰ vg/kg, no more than about 1 x 10¹¹ vg/kg, no more than about 5 x 10¹¹ vg/kg, no more than about 1 x 10¹² vg/kg, no more than about 5 x 10¹² vg/kg, no more than about 1 x 10¹³ vg/kg, no more than about 5 x 10¹³, or no more than about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered to a subject at a dose of about 1 x 10¹² vg/kg. In an aspect, a disclosed vector can be administered to a subject at a dose of about 1 x 10¹¹ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹² vg per subj ect total to about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total. In an aspect, a disclosed vector can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.

[0315] In an aspect, a disclosed method of cleaving surface-bound IgM can further comprise administering to the subject a therapeutically effective amount of a therapeutic agent. A therapeutic agent can be any disclosed agent that effects a desired clinical outcome.

[0316] In an aspect, a disclosed method of cleaving surface-bound IgM can further comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, the method can further comprise continuing to treat the subject. In an aspect, in the presence of adverse effects, the method can further comprise modifying the treating step. Methods of monitoring a subject’s well-being can include both subjective and objective criteria (and are discussed supra). Such methods are known to the skilled person.

[0317] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG can further comprise administering to the subject a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway. In an aspect, such an agent can comprise an enzyme for enzyme replacement therapy. In an aspect, a disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway. In an aspect, a disclosed method can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.

[0318] In an aspect, a disclosed method of cleaving surface-bound IgM can further comprise administering one or more immune modulators. In an aspect, a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, a disclosed immune modulator can be Tacrolimus. In an aspect, a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.1 mg/kg body weight to about 0.6 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary to achieve a desired clinical effect.

[0319] In an aspect, a disclosed immune modulator can be administered orally about one hour before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered orally about one hour or a few days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before or a few days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof.

[0320] In an aspect, a disclosed method of cleaving surface-bound IgM can further comprise administering one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib). In an aspect, a proteasome inhibitor can be an agent that acts on plasma cells (e.g., daratumumab). In an aspect, an agent that acts on a plasma cell can be melphalan hydrochloride, melphalan, pamidronate disodium, carmustine, carfilzomib, carmustine, cyclophosphamide, daratumumab, doxorubicin hydrochloride liposome, doxorubicin hydrochloride liposome, elotuzumab, melphalan hydrochloride, panobinostat, ixazomib citrate, carfilzomib, lenalidomide, melphalan, melphalan hydrochloride, plerixafor, ixazomib citrate, pamidronate disodium, panobinostat, plerixafor, pomalidomide, pomalidomide, lenalidomide, selinexor, thalidomide, thalidomide, bortezomib, selinexor, zoledronic acid, or zoledronic acid.

[0321] In an aspect, a disclosed method of cleaving surface-bound IgM can further comprise administering one or more proteasome inhibitors or agents that act on plasma cells prior to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells concurrently with administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells subsequent to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can further comprise administering one or more proteasome inhibitors more than 1 time. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors repeatedly over time.

[0322] In an aspect, a disclosed method of cleaving surface-bound IgM can further comprise administering one or more immunosuppressive agents. In an aspect, an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, anti -thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or a combination thereof. In an aspect, a disclosed method can comprise administering one or more immunosuppressive agents more than 1 time. In an aspect, a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time. In an aspect, a disclosed method can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.

[0323] In an aspect, a disclosed method of cleaving surface-bound IgM can further comprise administering a compound that exerts a therapeutic effect against B cells and/or a compound that targets or alters antigen presentation or humoral or cell mediated immune response. In an aspect, a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti-IGFIR antibody, a CD19 antibody (e.g., inebilizumab), an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof. Also disclosed herein are Treg infusions that can be administered as a way to help with immune tolerance (e.g., antigen specific Treg cells to AAV). [0324] In an aspect, a disclosed method can further comprise administering lipid nanoparticles (LNPs). In an aspect, LNPs can be organ-targeted. In an aspect, LNPs can be liver-targeted or testes-targeted. For example, in an aspect, mRNA therapy with LNP encapsulation for systemic delivery to a subject has the potential to restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzy me.

[0325] In an aspect, a disclosed method of cleaving surface-bound IgM can further comprise plasmapheresis and immunosuppression. In an aspect, a disclosed method can comprise using immunosuppression to decrease the T cell, B cell, and /or plasma cell population, decrease the innate immune response, inflammatory response, and antibody levels in general. [0326] In an aspect a disclosed method can comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed enzyme, a disclosed recombinant enzy me, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells and/or a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response.

[0327] In an aspect, a disclosed method can comprise modifying one or more of the disclosed steps. For example, modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject, or by changing the duration of time one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered to a subject.

[0328] In an aspect, a disclosed method can be altered by changing the amount of one or more disclosed enzymes or disclosed recombinant enzymes, one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.

[0329] In as aspect, a disclosed method can comprise concurrent administration of one or more of the following: one or more disclosed enzy mes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, one or more disclosed therapeutic agents, one or more disclosed immune modulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressive agents, one or more disclosed compounds that exert therapeutic effect against B cells, one or more disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response, or any combination thereof.

[0330] In an aspect, a disclosed immune modulator can be administered prior to or after the administration of a disclosed therapeutic agent.

[0331] In an aspect, a disclosed method of cleaving surface-bound IgM can further comprise generating one or more disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. For example, in an aspect, a disclosed method of cleaving surface-bound IgM can further comprise generating a disclosed viral or non- viral vector. In an aspect, generating a disclosed viral vector can comprise generating an AAV vector or a recombinant AAV (such as those disclosed herein).

[0332] In an aspect, a disclosed method of cleaving surface-bound IgM can further comprise validating one or more disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof .

[0333] In an aspect, a disclosed method of cleaving surface-bound IgM can further comprise gene editing one or more relevant genes (such as, for example, a missing, deficient, and/or mutant protein or enzy me), wherein editing includes but is not limited to single gene knockout, loss of function screening of multiple genes at one, gene knockin, or a combination thereof.

[0334] In an aspect, a disclosed method of cleaving surface-bound IgM can further comprise administering an oligonucleotide therapeutic agent. A disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, noncoding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof. In an aspect, a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise a CRISPR-based endonuclease. In an aspect, a disclosed endonuclease can be Cas9. In an aspect, a disclosed Cas9 can be from Staphylococcus aureus or Streptococcus pyogenes.

[0335] In an aspect of a disclosed method, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered one or more times. In an aspect, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered for the duration of the subject’s life, or for the duration of the subject's treatment with, for example, gene therapy, enzy me replacement therapy, protein replacement therapy, and/or recombinant product. In an aspect, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered every' 6 weeks, every' 2 months, every' 3 months, every' 6 months, every' year, year other year, every 3 years, every 5 years, or for any other period deemed necessary by a skilled clinician.

[0336] In an aspect of a disclosed method, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered prior to, concurrent, or after the delivery and/or administration of enzyme replacement therapy, protein replacement, gene therapy, recombinant product, or any combination thereof.

[0337] In an aspect, a disclosed method of cleaving surface bound IgM can further comprise reducing and/or minimizing vector-mediated immunotoxicity' and/or transgene immunogenicity' (e.g., the ability to induce specific immunity). In an aspect, vector-mediated immunotoxicity' and/or transgene immunogenicity can decrease and/or reduce the efficacy of the recombinant product encoded by the transgene. In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability⁷ and/or likelihood of re-dosing a subject with one or more disclosed enzymes or recombinant enzy mes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subject with gene therapy, enzyme replacement therapy, protein replacement, or any combination thereof.

[0338] In an aspect of a disclosed method of cleaving surface bound IgM, IgM can be cleaved in the heavy chain at the interdomain region between constant domains Cp2 and Cp3. In an aspect, IgM can be cleaved between position 350 and position 365 of SEQ ID NO: 105. In an aspect, SEQ ID NO: 105 can represent the linear sequence of an IgM (for example, the substrate). In an aspect, IgM can be cleaved between positions 355 through 360 of IgM. In an aspect, IgM can be cleaved at SEQ ID NO: 1 10. In an aspect, IgM can be cleaved at the sequence comprising 355D, 356T, 357A, 3581, 359R, and 360V. In an aspect, IgM can be cleaved at position 355 through position 360 of the IgM of SEQ ID NO: 105. In an aspect, IgG can be cleaved below the hinge region, thereby yielding F(ab’)2 and Fc fragments. In an aspect, IgG can be cleaved to separate the Cp2 and Cp3 domains from the Cpl domain. In an aspect, IgG can be cleaved at SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or any combination thereof. In an aspect, IgG can be cleaved at SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof. In an aspect, IgG can be cleaved at positions 109 through 124 of the IgG of SEQ ID NO: 106. positions 106 through 120 of the IgG of SEQ ID NO: 107. positions 156 through 171 ofthe lgG of SEQ ID NO: 108, positions 106 through 121 of lgG of SEQ ID NO: 109, or any combination thereof. In an aspect, IgG can be cleaved at positions 116 through 122 of the IgG of SEQ ID NO: 106, positions 112 through 118 of the IgG of SEQ ID NO: 107, positions 163 through 169 ofthe lgG of SEQ ID NO: 108, positions 113 through 119 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, IgG can be cleaved SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO:118, or any combination thereof.

[0339] Disclosed herein is a method of cleaving immunoglobulin M (IgM) bound to the surface of B cells the method comprising contacting one or more IgM molecules with one or more disclosed recombinant enzymes and one or more disclosed recombinant enzymes comprising the sequence of any one SEQ ID NO: 119 - SEQ ID NO: 159.

[0340] Disclosed herein is a method of cleaving immunoglobulin M (IgM) bound to the surface of B cells the method comprising contacting one or more IgM molecules with one or more disclosed recombinant enzymes and one or more disclosed recombinant enzymes comprising the sequence of any one SEQ ID NO: 119 - SEQ ID NO: 159.

[0341] Disclosed herein is a method of cleaving immunoglobulin M (IgM) bound to the surface of B cells the method comprising contacting one or more IgM molecules with one or more disclosed recombinant enzymes and one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 1 19.

[0342] Disclosed herein is a method of cleaving immunoglobulin M (IgM) bound to the surface of B cells the method comprising contacting one or more IgM molecules with one or more disclosed recombinant enzymes and one or more disclosed recombinant enzymes comprising the sequence of any one SEQ ID NO: 119 - SEQ ID NO: 159.

[0343] Disclosed herein is a method of cleaving immunoglobulin M (IgM) bound to the surface of B cells the method comprising contacting one or more IgM molecules with one or more disclosed recombinant enzymes and one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 119.

[0344] In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of SEQ ID NO: 119. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. In an aspect a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%. at least 80%. at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 1 19. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159.

[0345] In an aspect, a disclosed method can further comprise administering one or more times one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0346] In an aspect, a disclosed method can further comprise measuring and/or determining a subject’s pre-treatment level of circulating IgG and/or IgM and/or a subject’s pre-treatment level of surface bound IgM. In an aspect, a disclosed method can further comprise measuring and/or determining one or more times a subject’s level of circulating IgG and/or IgM and/or a subject’s level of surface bound IgM (e.g., before, during, and after a disclosed administering step).

[0347] In an aspect, a disclosed method can be performed using a singular disclosed enzyme having both IgG and IgM specific protease activity. In an aspect, a disclosed method can be performed using a disclosed enzyme having IgG specific protease activity and a disclosed enzyme having IgM specific protease activity.

E. Methods of Reducing Circulating Levels of IgM and/or IgG

[0348] Disclosed herein is a method of reducing the level of circulating intact IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed enzymes or disclosed recombinant enzy mes. Disclosed herein is a method of reducing the level of circulating intact IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed nucleic acid molecules. Disclosed herein is a method of reducing the level of circulating intact IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed vectors. Disclosed herein is a method of reducing the level of circulating intact IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed pharmaceutical formulations. Disclosed herein is a method of reducing the level of circulating intact IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO: 15. Disclosed herein is a method of reducing the level of circulating intact IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:38. Disclosed herein is a method of reducing the level of circulating intact IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzy me comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03. Disclosed herein is a method of reducing the level of circulating intact IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzy me comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06. Disclosed herein is a method of reducing the level of circulating intact IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enz me comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09. Disclosed herein is a method of reducing the level of circulating intact IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzy me comprising the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. Disclosed herein is a method of reducing the level of circulating intact IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically^ effective amount of an enzy me comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO:15.

[0349] Disclosed herein is a method of reducing the level of circulating intact IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO:47 - SEQ ID NO:61 or SEQ ID NO:70 - SEQ ID NO:84. Disclosed herein is a method of reducing the level of circulating intact IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, or SEQ ID NO:61. Disclosed herein is a method of reducing the level of circulating intact IgM in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, or SEQ ID NO:84.

[0350] Disclosed herein is a method reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed enzymes or disclosed recombinant enzymes. Disclosed herein is a method of reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed nucleic acid molecules. Disclosed herein is a method of reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed vectors. Disclosed herein is a method of reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed pharmaceutical formulations.

[0351] Disclosed herein is a method of reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in any one of SEQ ID NO: 16 - SEQ ID NO:23. Disclosed herein is a method of reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in any one of SEQ ID NO:39 - SEQ ID NO:46. Disclosed herein is a method of reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein is a method of reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21 . Disclosed herein is a method of reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23.

[0352] Disclosed herein is a method of reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO: 62 - SEQ ID NO: 69 or SEQ ID NO: 85 - SEQ ID NO: 92. Disclosed herein is a method of reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:63, SEQ ID NO:64. SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO 69. Disclosed herein is a method of reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90. SEQ ID NO:91, or SEQ ID NO:92.

[0353] Disclosed herein is a method reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG specific protease activity', and (ii) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgM specific protease activity.

[0354] Disclosed herein is a method reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG specific protease activity (such as, for example, the enzyme of SEQ ID NO:08, SEQ ID NO:09, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 15). and (ii) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgM specific protease activity⁷ (such as, for example, the enzyme of any one of SEQ ID NO: 119 - SEQ ID NO: 159).

[0355] Disclosed herein is a method of reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO:47 - SEQ ID NO:61 and (ii) a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161.

[0356] Disclosed herein is a method of reducing the level of circulating intact IgM and IgG in the serum of a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of the recombinant enzyme of SEQ ID NO:08, SEQ ID NO:09, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 14, or SEQ ID NO: 15, and (ii) a therapeutically effective amount of the recombinant enzyme of SEQ ID NO: 119, SEQ ID NO: 120, or SEQ ID NO: 121.

[0357] Disclosed herein is a method of reducing the level of circulating intact IgG in the serum of a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. [0358] In an aspect, a disclosed IgM molecule can be neutralizing against an exogenously administered protein. In an aspect, a disclosed IgM molecule can be neutralizing against enzyme replacement therapy, protein replacement therapy , and/or recombinant product. In an aspect, a disclosed IgM molecule can be neutralizing against a non-viral vector or a viral vector. In an aspect, a disclosed IgM molecule can be neutralizing against a protein associated with a non-viral vector or a viral vector. For example, in an aspect, a disclosed IgM molecule can be neutralizing against a viral vector capsid protein (e.g., an AAV capsid protein).

[0359] In an aspect, a disclosed method can further comprise diagnosing a subject as being in need of one or more disclosed enzymes, one or more disclosed recombinant enzymes, one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0360] In an aspect of a disclosed method, there is at least a 10% reduction, at least a 20% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, at least an 80% reduction, at least a 90% reduction, or at least a 99% reduction in the level of circulating intact IgM and/or intact IgG.

[0361] In an aspect, a disclosed IgM molecule can be neutralizing against one or more transplanted organs. In an aspect, a disclosed method can degrade one or more anti-HLA antibodies directed at the one or more transplanted organs.

[0362] 'In an aspect, a subject can have a high sustained antibody titer (HSAT - defined as titers of > 12,800). In an aspect, a subject can have a high sustained antibody titer (HSAT) against one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more non-viral vectors or viral vectors, one or more proteins associated with a non-viral vector, one or more proteins associated with a viral vector, or any combination thereof. In an aspect, after the contacting step, a disclosed HSAT can be reduced and/or eliminated. In an aspect, after the reduction and/or elimination of one or more HSATs, a subject can be re-dosed. In an aspect, after the reduction and/or elimination of one or more HSATs, a subject can be re-dosed with one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more recombinant products, one or more non-viral vectors or viral vectors, one or more proteins associated with a non-viral vector, one or more proteins associated with a viral vector, or any combination thereof.

[0363] In an aspect, a disclosed method can further comprise measuring one or more time the levels of IgM and/or IgG in the subject (such as, for example, in the subject’s sera).

[0364] In an aspect, a disclosed method of reducing circulating levels of IgM and/or IgG and IgM can clear and/or remove IgM and/or IgG and IgM from circulation in a subject. In an aspect, a disclosed method can cleave and/or degrade circulating IgM and/or IgG and IgM in a subject. In an aspect, a disclosed method can cleave and/or degrade all or nearly all circulating IgM and/or IgG and IgM in a subject. In an aspect, a disclosed method can transiently clear and/or remove IgM and/or IgG and IgM from circulation in a subject, or can transiently cleave and/or degrade circulating IgM and/or IgG and IgM in a subject. In an aspect, a disclosed method can completely or nearly completely clear and/or remove IgM and/or IgG and IgM from circulation in a subject. In an aspect, a disclosed method can reduce and/or diminish the level of circulating IgM and/or IgG and IgM in a subject, or can transiently reduce and/or diminish the level of circulating IgM and/or IgG and IgM in a subject. In aspect, a disclosed transient reduction and/or diminishment can comprise about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[0365] In an aspect, a disclosed method of reducing levels of circulating intact IgM or circulating intact IgM and IgG can block and/or minimize activation of the complement cascade. In an aspect, a disclosed method of reducing levels of circulating intact IgM or circulating intact IgM and IgG can block and/or minimize activation of the formation of the Cl complex and/or C3 complex. In an aspect, a disclosed method of reducing levels of circulating intact IgM or circulating intact IgM and IgG can block and/or minimize activation of the classical complement pathway. In an aspect, a disclosed method of reducing the level of circulating intact IgM or circulating intact IgM and IgG can transiently block and/or minimize activation of the complement cascade, can transiently block and/or minimize activation of the formation of the Cl complex and/or C3 complex, can transiently block and/or minimize activation of the classical complement pathway, or any combination thereof. In an aspect, a disclosed method of reducing levels of circulating intact IgM or circulating intact IgM and IgG can reduce and/or minimize vector mediated immunotoxicity in a subject. In an aspect, a disclosed method of reducing levels of circulating intact IgM or circulating intact IgM and IgG can enable and/or allow vector re-dosing in a subject. In an aspect, a disclosed method of reducing levels of circulating intact IgM or circulating intact IgM and IgG can enable and/or allow re-dosing of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof in a subject.

[0366] In an aspect, a disclosed method of reducing levels of circulating intact IgM or circulating intact IgM and IgG can remove and/or cleave surface-bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells. In an aspect, the disclosed removal and/or cleavage of IgM from the surface of B cells can be reversible and/or transient. In an aspect, a disclosed method of reducing levels of circulating intact IgM or circulating intact IgM and IgG can transiently remove and/or cleave surface-bound IgM from B cells and/or can transiently remove and/or cleave IgM from the surface of B cells.

[0367] In an aspect, a disclosed method of reducing the level of circulating intact IgM or circulating intact IgM and IgG can reduce and/or minimize transgene immunogenicity in a subject. In an aspect, a disclosed method of reducing the level of circulating intact IgM or circulating intact IgM and IgG can improve and/or enhance transgene efficacy and/or expression in a subject. In an aspect, improving and/or enhancing transgene efficacy and/or expression in a subject can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of an improvement and/or enhancement when compared to a pre-existing level of transgene efficacy and/or expression such as, for example, a pre-treatment level (prior to administering one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, the amount of improvement and/or enhancement can be 10-20%, 20-30%, 30-40%. 40-50%, 50-60%, 60-70%, 70-80%. 80-90%, or 90-100% more than a pre-existing level such as a pre-treatment level (prior to administering one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, an improvement and/or enhancement can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having received or not having been administered one or more disclosed enzy mes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, an improvement and/or enhancement can be a partial or incomplete restoration. In an aspect, an improvement and/or enhancement can be complete or near complete such that the level of neutralizing antibodies or neutralizing IgG and/or IgM molecules is similar to that of a wildtype or control level.

[0368] In an aspect, a disclosed method reducing the level of circulating intact IgM or circulating intact IgM and IgG can modulate humoral immunity in a subject. In an aspect, modulating humoral immunity⁷ in a subject can be an increase in the functionality⁷ of the subject’s humoral immunity⁷, or can be a decrease in the functional of the subject’s humoral immunity.

[0369] In an aspect of a disclosed method, intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an administered gene therapy. In an aspect of a disclosed method, intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an administered therapeutic protein or a therapeutic recombinant protein. In an aspect of a disclosed method, the intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an encoded therapeutic protein or encoded therapeutic recombinant protein.

[0370] In an aspect, a disclosed method can comprise administering to a subject in need thereof a theoretically effectively amount of one or more disclosed enzymes or disclosed recombinant enzymes, one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0371] In an aspect, a subject can have or can be suspected of having one of more autoimmune diseases and/or disorders, one or more autoinfl ammatory diseases and/or disorders, one or more inflammatory’ diseases and/or disorders, or any combination thereof. Disclosed autoimmune diseases and/or disorders are known to the art and discussed supra. Disclosed autoinflammatory diseases and/or disorders are known to the art and discussed supra. Disclosed inflammatory' diseases and/or disorders are known to the art and discussed supra.

[0372] In an aspect, a subject can be in need of one or more organ transplants. In an aspect, a subj ect can have received one or more organ transplants. In an aspect, a disclosed organ transplant can comprise the heart, one or both kidneys, the liver, one or both lungs, the pancreas, part or all of the colon or the intestines, or any combination thereof.

[0373] In an aspect, a subject can be in need of gene therapy, can have received gene therapy, or can be receiving gene therapy. In an aspect, a subject can be treated with gene therapy or can have a disease and/or disorder that can be treated with gene therapy. In an aspect, a subject can have or be suspected of having a disclosed disease or disorder that can be treated with gene therapy.

[0374] In an aspect, a disclosed gene therapy can comprise the delivery and/or repair of one or more disclosed genes. In an aspect, a disclosed gene therapy can comprise AAV-based gene therapy, Adenovirus-based gene therapy, Lentiviral gene therapy, or the combination thereof. In an aspect, a disclosed gene therapy can comprise the delivery' and/or repair of one or more disclosed genes. In an aspect, a disclosed gene therapy can comprise AAV-based gene therapy, Adenovirus-based gene therapy. Lentiviral gene therapy, or the combination thereof.

[0375] Genetic diseases and/or disorders or diseases and/or disorders that can be treated with gene therapy are known to the art and are discussed supra. In an aspect, a disclosed gene therapy can comprise the delivery and/or repair of one or more disclosed genes. Genetic diseases and/or disorders can be due to a defect in one or more genes known to the art and discussed supra.

[0376] In an aspect, a disclosed method of reducing the level of circulating intact IgM or circulating intact IgM and IgG can block and/or minimize activation of the complement cascade. In an aspect, a disclosed method of reducing the level of circulating intact IgM or circulating intact IgM and IgG can block and/or minimize activation of the formation of the Cl complex and/or C3 complex. In an aspect, a disclosed method of reducing the level of circulating intact IgM or circulating intact IgM and IgG can block and/or minimize activation of the classical complement pathway. In an aspect, a disclosed method of reducing the level of circulating intact IgM or circulating intact IgM and IgG can transiently block and/or minimize activation of the complement cascade, can transiently block and/or minimize activation of the formation of the Cl complex and/or C3 complex, can transiently block and/or minimize activation of the classical complement pathway, or any combination thereof.

[0377] In an aspect, a disclosed method of reducing circulating levels of IgG or circulating intact IgM and IgG can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme delivered by gene therapy. In an aspect, a disclosed method of reducing circulating levels of IgG or circulating intact IgM and IgG can comprise restoring one or more aspects of cellular homeostasis and/or cellular functionality' and/or metabolic dysregulation. In an aspect, restoring one or more aspect of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation comprises restoring the functionality' and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0378] In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.

[0379] In an aspect, restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.

[0380] In an aspect of a disclosed method, techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are discussed supra.

[0381] In an aspect of a disclosed method, contacting a cell can comprising methods known to the art. For example, contacting can comprise administering to a subject one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof .

[0382] In an aspect, administering can comprise intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intra-CSF, intrathecal, intraventricular, intrahepatic, hepatic intraarterial, hepatic portal vein (HPV), or in utero administration. In an aspect, a disclosed composition, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be administered via intra-CSF administration in combination with RNAi, antisense oligonucleotides, miRNA, one or more small molecules, one or more therapeutic agents, one or more proteasome inhibitors, one or more immune modulators, and/or a gene editing system. In an aspect, a disclosed composition, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be administered via LNP administration. In an aspect, a disclosed composition, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be concurrently and/or serially administered to a subject via multiple routes of administration. For example, in an aspect, administering a disclosed nucleic acid molecule, a disclosed vector, and/or a disclosed pharmaceutical formulation can comprise intravenous administration and intra-cistem magna (ICM) administration. In an aspect, administering a disclosed composition, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can comprise IV administration and intrathecal (ITH) administration. In an aspect, a disclosed method can employ multiple routes of administration to the subject. In an aspect, a disclosed method can employ a first route of administration that can be the same or different as a second and/or subsequent routes of administration.

[0383] In an aspect, a therapeutically effective amount of a disclosed enzyme or disclosed recombinant enzyme can comprise about 0.01 mg/kg body weight to about 100 mg/kg body weight. In an aspect, a disclosed enzyme or disclosed recombinant enzyme can be therapeutically effective when the dose comprises about 0.01 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg. about 75 mg/kg. about 80 mg/kg. about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg body weight.

[0384] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG, a therapeutically effective amount of disclosed vector can be delivered to a subject via intravenous (IV) administration and can comprise a range of about 1 x 10¹⁰ vg/kg to about 2 x 10¹⁴ vg/kg. In an aspect, for example, a disclosed vector can be administered at a dose of about 1 x 10¹¹ vg/kg to about 8 x 10¹³ vg/kg or about 1 x 10¹² vg/kg to about 8 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 10¹³ vg/kg to about 6 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of at least about 1 x 10¹⁰ vg/kg, at least about 5 x IO¹⁰ vg/kg, at least about 1 x 10¹¹ vg/kg, at least about 5 x 10¹¹ vg/kg, at least about 1 x 10¹² vg/kg, at least about 5 x 10¹² vg/kg, at least about 1 x 10¹³ vg/kg, at least about 5 x 10¹³ vg/kg, or at least about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered at a dose of no more than about 1 x 10¹⁰ vg/kg, no more than about 5 x 10¹⁰ vg/kg. no more than about 1 x 10¹¹ vg/kg, no more than about 5 x 10¹¹ vg/kg. no more than about 1 x 10¹² vg/kg, no more than about 5 x 10¹² vg/kg, no more than about 1 x 10¹³ vg/kg, no more than about 5 x 10¹³, or no more than about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered to a subject at a dose of about 1 x 10¹² vg/kg. In an aspect, a disclosed vector can be administered to a subject at a dose of about 1 x 10¹¹ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total. In an aspect, a disclosed vector can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.

[0385] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG can further comprise administering to the subject a therapeutically effective amount of a therapeutic agent. A therapeutic agent can be any disclosed agent that effects a desired clinical outcome. [0386] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG can further comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, the method can further comprise continuing to treat the subject. In an aspect, in the presence of adverse effects, the method can further comprise modifying the treating step. Methods of monitoring a subject’s well-being can include both subjective and objective criteria (and are discussed supra). Such methods are known to the skilled person.

[0387] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG can further comprise administering to the subject a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway. In an aspect, such an agent can comprise an enzyme for enzyme replacement therapy. In an aspect, a disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzy matic pathway. In an aspect, a disclosed method can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.

[0388] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG can further comprise administering one or more immune modulators. In an aspect, a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, a disclosed immune modulator can be Tacrolimus. In an aspect, a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.1 mg/kg body weight to about 0.6 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body w eight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary to achieve a desired clinical effect.

[0389] In an aspect, a disclosed immune modulator can be administered orally about one hour before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered orally about one hour or a few' days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before or a few days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof.

[0390] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG can further comprise administering one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib). In an aspect, a proteasome inhibitor can be an agent that acts on plasma cells (e.g., daratumumab). In an aspect, an agent that acts on a plasma cell can be melphalan hydrochloride, melphalan, pamidronate disodium, carmustine, carfilzomib, carmustine, cyclophosphamide, daratumumab, doxorubicin hydrochloride liposome, doxorubicin hydrochloride liposome, elotuzumab, melphalan hydrochloride, panobinostat. ixazomib citrate, carfilzomib, lenalidomide, melphalan, melphalan hydrochloride, plerixafor, ixazomib citrate, pamidronate disodium, panobinostat, plerixafor, pomalidomide, pomalidomide, lenalidomide, selinexor, thalidomide, thalidomide, bortezomib, selinexor, zoledronic acid, or zoledronic acid.

[0391] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG can further comprise administering one or more proteasome inhibitors or agents that act on plasma cells prior to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells concurrently with administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells subsequent to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can further comprise administering one or more proteasome inhibitors more than 1 time. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors repeatedly over time.

[0392] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG can further comprise administering one or more immunosuppressive agents. In an aspect, an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, anti -thymocyte globulin (ATG), cyclosporine (CSP), my cophenolate mofetil (MMF), steroids, or a combination thereof. In an aspect, a disclosed method can comprise administering one or more immunosuppressive agents more than 1 time. In an aspect, a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time. In an aspect, a disclosed method can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.

[0393] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG can further comprise administering a compound that exerts a therapeutic effect against B cells and/or a compound that targets or alters antigen presentation or humoral or cell mediated immune response. In an aspect, a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti-IGFIR antibody, a CD 19 antibody (e.g.. inebilizumab). an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof. Also disclosed herein are Treg infusions that can be administered as a way to help with immune tolerance (e.g., antigen specific Treg cells to AAV).

[0394] In an aspect, a disclosed method can further comprise administering lipid nanoparticles (LNPs). In an aspect. LNPs can be organ-targeted. In an aspect, LNPs can be liver-targeted or testes-targeted. For example, in an aspect, mRNA therapy with LNP encapsulation for systemic delivery to a subject has the potential to restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0395] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG can further comprise plasmapheresis and immunosuppression. In an aspect, a disclosed method can comprise using immunosuppression to decrease the T cell, B cell, and /or plasma cell population, decrease the innate immune response, inflammatory response, and antibody levels in general.

[0396] In an aspect, a disclosed method can comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells and/or a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response.

[0397] In an aspect, a disclosed method can comprise modifying one or more of the disclosed steps. For example, modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more disclosed enzy mes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject, or by changing the duration of time one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered to a subject.

[0398] In an aspect, a disclosed method can be altered by changing the amount of one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.

[0399] In as aspect, a disclosed method can comprise concurrent administration of one or more of the following: one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, one or more disclosed therapeutic agents, one or more disclosed immune modulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressive agents, one or more disclosed compounds that exert therapeutic effect against B cells, one or more disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response, or any combination thereof.

[0400] In an aspect, a disclosed immune modulator can be administered prior to or after the administration of a disclosed therapeutic agent.

[0401] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG can further comprise generating one or more disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. [0402] For example, in an aspect, a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG can further comprise generating a disclosed viral or non-viral vector. In an aspect, generating a disclosed viral vector can comprise generating an AAV vector or a recombinant AAV (such as those disclosed herein). [0403] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG, can further comprise gene editing one or more relevant genes (such as, for example, a missing, deficient, and/or mutant protein or enzyme), wherein editing includes but is not limited to single gene knockout, loss of function screening of multiple genes at one, gene knockin, or a combination thereof.

[0404] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG, can further comprise administering an oligonucleotide therapeutic agent. A disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof. In an aspect, a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise a CRISPR-based endonuclease. In an aspect, a disclosed endonuclease can be Cas9. In an aspect, a disclosed Cas9 can be from Staphylococcus aureus or Streptococcus pyogenes.

[0405] In an aspect of a disclosed method of reducing circulating levels of IgM or circulating intact IgM and IgG, can further comprise generating and/or validating one or more disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0406] In an aspect of a disclosed method, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered one or more times. In an aspect, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered for the duration of the subject’s life, or for the duration of the subject’s treatment with, for example, gene therapy, enzyme replacement therapy, protein replacement therapy, and/or recombinant product. In an aspect, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered every 6 weeks, every’ 2 months, every 3 months, every 6 months, every year, year other year, every 3 years, every 5 years, or for any other period deemed necessary by a skilled clinician.

[0407] In an aspect of a disclosed method, a disclosed enzy me, a disclosed recombinant enzy me, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered prior to, concurrent, or after the delivery and/or administration of enzyme replacement therapy, protein replacement, gene therapy, a recombinant product, or any combination thereof.

[0408] In an aspect, a disclosed method of circulating levels of IgM and/or IgG can further comprise reducing and/or minimizing vector-mediated immunotoxicity and/or transgene immunogenicity (e.g., the ability to induce specific immunity). In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the efficacy of the recombinant product encoded by the transgene. In an aspect, vector-mediated immunotoxicity and/or trans gene immunogenicity can decrease and/or reduce the ability and/or likelihood of redosing a subject with one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subject with gene therapy, enzyme replacement therapy, protein replacement, or any combination thereof.

[0409] In an aspect of a disclosed method of inhibiting complement, IgM can be cleaved in the heavy' chain at the interdomain region between constant domains Cp2 and Cp3. In an aspect, IgM can be cleaved between position 350 and position 365 of SEQ ID NO: 105. In an aspect, SEQ ID NO: 105 can represent the linear sequence of an IgM (for example, the substrate). In an aspect, IgM can be cleaved between positions 355 through 360 of IgM. In an aspect, IgM can be cleaved at SEQ ID NO: 110. In an aspect, IgM can be cleaved at the sequence comprising 355D, 356T, 357A, 3581, 359R, and 360V. In an aspect, IgM can be cleaved at position 355 through position 360 of the IgM of SEQ ID NO: 105. In an aspect, IgG can be cleaved below the hinge region, thereby yielding F(ab’)2 and Fc fragments. In an aspect, IgG can be cleaved to separate the CLI2 and Cp3 domains from the Cpl domain. In an aspect, IgG can be cleaved at SEQ ID NO:111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114. or any combination thereof. In an aspect, IgG can be cleaved at SEQ ID NO: 115. SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118. or any combination thereof. In an aspect, IgG can be cleaved at positions 109 through 124 of the IgG of SEQ ID NO: 106, positions 106 through 120 of the IgG of SEQ ID NO: 107, positions 156 through 171 of the IgG of SEQ ID NO: 108, positions 106 through 121 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, IgG can be cleaved at positions 116 through 122 of the IgG of SEQ ID NO: 106, positions 112 through 118 of the IgG of SEQ ID NO: 107, positions 163 through 169 ofthe lgG of SEQ ID NO: 108, positions 113 through 119 of IgG of SEQ ID NO:109, or any combination thereof. In an aspect, IgG can be cleaved SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof. [0410] Disclosed herein is method of reducing the level of circulating intact IgM and/or IgG the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzy mes having IgM specific protease activity' and one or more disclosed enzymes having IgG specific protease activity.

[0411] Disclosed herein is method of reducing the level of circulating intact IgM and/or IgG the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes having IgM specific protease activity' and one or more disclosed recombinant enzy mes comprising the sequence of any one SEQ ID NO: 119 - SEQ ID NO: 159.

[0412] Disclosed herein is method of reducing the level of circulating intact IgM and/or IgG the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes comprising the sequence of any one of SEQ ID NO: 01 - SEQ ID NO:46 and one or more disclosed recombinant enzymes comprising the sequence of any one SEQ ID NO: 119 - SEQ ID NO: 159.

[0413] Disclosed herein is method of reducing the level of circulating intact IgM and/or IgG the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes having IgM specific protease activity' and one or more disclosed recombinant enzy mes comprising the sequence of SEQ ID NO: 119.

[0414] Disclosed herein is method of reducing the level of circulating intact IgM and/or IgG the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes having IgM specific protease activity and one or more disclosed recombinant enzy mes comprising the sequence of any one SEQ ID NO: 119 - SEQ ID NO: 159.

[0415] Disclosed herein is method of reducing the level of circulating intact IgM and/or IgG the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 11 or SEQ ID NO: 12 and one or more disclosed recombinant enzy mes comprising the sequence of SEQ ID NO: 119.

[0416] Disclosed herein is method of reducing the level of circulating intact IgM and/or IgG the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 14 or SEQ ID NO: 15 and one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 119.

[0417] In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of SEQ ID NO: 119. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159. In an aspect a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzy me comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 119. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity' to the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a sequence having at least 50%. at least 60%. at least 70%. at least 80%, at least 90%, or more than 90% identity to the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159.

[0418] In an aspect, a disclosed method can further comprise administering one or more times one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0419] In an aspect, a disclosed method can further comprise measuring and/or determining a subject’s pre-treatment level of circulating IgG and/or IgM and/or a subject’s pre-treatment level of surface bound IgM. In an aspect, a disclosed method can further comprise measuring and/or determining one or more times a subject’s level of circulating IgG and/or IgM and/or a subject’s level of surface bound IgM (e.g., before, during, and after a disclosed administering step).

[0420] In an aspect, a disclosed method can be performed using a singular disclosed enzyme having both IgG and IgM specific protease activity. In an aspect, a disclosed method can be performed using a disclosed enzyme having IgG specific protease activity and a disclosed enzyme having IgM specific protease activity.

F. Methods of Inhibiting Complement Activation

[0421] Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed enzymes or disclosed recombinant enzymes. Disclosed herein is a method of inhibiting complement activation in a subj ect, the method comprising administering to the subj ect in need thereof a therapeutically effective amount of one or more disclosed nucleic acid molecules. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed vectors. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed pharmaceutical formulations. [0422] Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO: 15. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:38.

[0423] Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO:08 or SEQ ID NO:09. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15.

[0424] Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO:47 - SEQ ID NO:61 or SEQ ID NO:70 - SEQ ID NO:84. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, or SEQ ID NO:61 . Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:71. SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, or SEQ ID NO:84.

[0425] Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG specific protease activity, and (ii) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgM specific protease activity.

[0426] Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG specific protease activity (such as, for example, the enzyme of SEQ ID NO:08, SEQ ID NO:09, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 15), and (ii) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgM specific protease activity (such as, for example, the enzyme of any one of SEQ ID NO: 119 - SEQ ID NO: 159).

[0427] Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG specific protease activity’ (such as, for example, the enzyme of SEQ ID NO: 11. SEQ ID NO: 12, SEQ ID NO: 14. or SEQ ID NO: 15), and (ii) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgM specific protease activity' (such as, for example, the enzyme of SEQ ID NO: 119).

[0428] Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO:47 - SEQ ID NO:61 and (ii) a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO:161.

[0429] Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of the recombinant enzyme of SEQ ID NO:08, SEQ ID NO:09, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 15, and (ii) a therapeutically effective amount of the recombinant enzyme of SEQ ID NO: 119, SEQ ID NO: 120. or SEQ ID NO: 121.

[0430] Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. [0431] Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed enzymes or disclosed recombinant enzymes. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed nucleic acid molecules.

Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed vectors. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed pharmaceutical formulations.

[0432] Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in any one of SEQ ID NO: 16 - SEQ ID NO:23. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in any one of SEQ ID NO:39 - SEQ ID NO:46. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO:22 or SEQ ID NO:23.

[0433] Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO:62 - SEQ ID NO:69 or SEQ ID NO:85 - SEQ ID NO:92. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69. Disclosed herein is a method of inhibiting complement activation in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, or SEQ ID NO:92.

[0434] In an aspect of a disclosed method of inhibiting complement activation, the formation of the C 1 complex and/or C3 complex can be blocked and/or minimized. In an aspect of a disclosed method of inhibiting complement activation, the activation of the classical complement pathway can be blocked and/or minimized. In an aspect, a disclosed method can transiently block and/or minimize activation of the complement cascade, can transiently block and/or minimize activation of the formation of the Cl complex and/or C3 complex, can transiently block and/or minimize activation of the classical complement pathway, or any combination thereof. In an aspect, a disclosed method of inhibiting complement activation can further comprise clearing and/or removing IgM from circulation in a subject. In an aspect, a disclosed method of inhibiting complement activation can further comprise cleaving and/or degrading circulating IgM in a subj ect. In an aspect, a disclosed method of inhibiting complement activation can further comprise cleaving and/or degrading all or nearly all circulating IgM in a subject. In an aspect, a disclosed method of inhibiting complement activation can further comprise transiently clearing and/or removing IgM from circulation in a subject, or transiently clearing and/or degrading circulating IgM in a subject. In an aspect, a disclosed method of inhibiting complement activation can further comprise completely or nearly completely clearing and/or removing IgM from circulation in a subj ect. In an aspect, a disclosed method of inhibiting complement activation can further comprise reducing and/or diminishing the level of circulating IgM in a subject, or transiently reducing and/or diminishing the level of circulating IgM in a subject.

[0435] In an aspect, a disclosed method of inhibiting complement activation can further comprise clearing and/or removing IgM and/or IgG and IgM from circulation in a subject. In an aspect, a disclosed method of inhibiting complement activation can further comprise clearing and/or degrade circulating IgM and/or IgG and IgM in a subject. In an aspect, a disclosed method of inhibiting complement activation can further comprise cleaving and/or degrading all or nearly all circulating IgM and/or IgG and IgM in a subject. In an aspect, a disclosed method of inhibiting complement activation can further comprise transiently clearing and/or removing IgM and/or IgG and IgM from circulation in a subject, or can transiently cleaving and/or degrading circulating IgM and/or IgG and IgM in a subject.

[0436] In an aspect, a disclosed method of inhibiting complement activation can further comprise completely or nearly completely clearing and/or removing IgM and/or IgG and IgM from circulation in a subject. In an aspect, a disclosed method of inhibiting complement activation can further comprise reducing and/or diminishing the level of circulating IgM and/or IgG and IgM in a subject, or can transiently reduce and/or diminish the level of circulating IgM and/or IgG and IgM in a subject. In aspect, a disclosed transient reduction and/or diminishment can comprise about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[0437] In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against an exogenously administered protein. In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against enzyme replacement therapy or protein replacement therapy or recombinant product. In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against a non-viral vector or a viral vector. In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against a protein associated with a non-viral vector or a viral vector. For example, in an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against a viral vector capsid protein (e.g., an AAV capsid protein).

[0438] In an aspect of a disclosed method, there is at least a 10% reduction, at least a 20% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, at least an 80% reduction, at least a 90% reduction, or at least a 99% reduction in the level of circulating intact IgM and/or intact IgG.

[0439] In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against one or more transplanted organs. In an aspect, a disclosed method can degrade one or more anti-HLA antibodies directed at the one or more transplanted organs.

[0440] In an aspect, a subject can have a high sustained antibody titer (HSAT - defined as titers of > 12,800). In an aspect, a subject can have a high sustained antibody titer (HSAT) against one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more non-viral vectors or viral vectors, one or more proteins associated with a non-viral vector, one or more proteins associated with a viral vector, or any combination thereof. In an aspect, after the contacting step, a disclosed HSAT can be reduced and/or eliminated. In an aspect, after the reduction and/or elimination of one or more HSATs, a subject can be re-dosed. In an aspect, after the reduction and/or elimination of one or more HSATs, a subject can be re-dosed with one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more recombinant products, one or more non-viral vectors or viral vectors, one or more proteins associated with a non-viral vector, one or more proteins associated with a viral vector, or any combination thereof.

[0441] In an aspect, a disclosed method can further comprise measuring one or more time the levels of IgM and/or IgG in the subject (such as, for example, in the subject’s sera).

[0442] In an aspect, a disclosed method of inhibiting complement activation can enable and/or allow vector re-dosing in a subject. In an aspect, a disclosed method of inhibiting complement activation can enable and/or allow re-dosing of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof in a subj ect. [0443] In an aspect, a disclosed method of inhibiting complement activation can further comprise reducing and/or minimizing vector mediated immunotoxicity in a subject. In an aspect, a disclosed method of inhibiting complement activation can reduce and/or minimize transgene immunogenicity in a subject.

[0444] In an aspect, a disclosed method of inhibiting complement activation can improve and/or enhance transgene efficacy and/or expression in a subject. In an aspect, improving and/or enhancing transgene efficacy and/or expression in a subject can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of an improvement and/or enhancement when compared to a pre-existing level of transgene efficacy and/or expression such as, for example, a pre-treatment level (prior to administering one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, the amount of improvement and/or enhancement can be 10-20%, 20-30%, 30-40%, 40- 50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as a pre-treatment level (prior to administering one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, an improvement and/or enhancement can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having received or not having been administered one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, an improvement and/or enhancement can be a partial or incomplete restoration. In an aspect, an improvement and/or enhancement can be complete or near complete such that the level of neutralizing antibodies or neutralizing IgG and/or IgM molecules is similar to that of a wild-type or control level.

[0445] In an aspect, a disclosed method inhibiting complement activation can remove and/or cleave surface-bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells. In an aspect, the disclosed removal and/or cleavage of IgM from the surface of B cells can be reversible and/or transient. In an aspect, a disclosed method of inhibiting complement activation can transiently remove and/or cleave surface-bound IgM from B cells and/or can transiently remove and/or cleave IgM from the surface of B cells. In an aspect, a disclosed method can remove and/or cleave surface bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells without killing and/or injuring the B cells.

[0446] In an aspect, a disclosed method of inhibiting complement activation can modulate humoral immunity in a subject. In an aspect, modulating humoral immunity in a subject can be an increase in the functionality of the subject’s humoral immunity, or can be a decrease in the functional of the subject’s humoral immunity.

[0447] In an aspect of a disclosed method, intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an administered gene therapy. In an aspect of a disclosed method, intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an administered therapeutic protein or a therapeutic recombinant protein. In an aspect of a disclosed method, the intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an encoded therapeutic protein or encoded therapeutic recombinant protein.

[0448] In an aspect, a disclosed method of inhibiting complement activation can comprise administering to a subject in need thereof a theoretically effectively amount of one or more disclosed enzymes or disclosed recombinant enzymes, one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0449] In an aspect, a subject can have or can be suspected of having one of more autoimmune diseases and/or disorders, one or more autoinfl ammatory diseases and/or disorders, one or more inflammatory' diseases and/or disorders, or any combination thereof. Disclosed autoimmune diseases and/or disorders are known to the art and discussed supra. Disclosed autoinflammatory diseases and/or disorders are known to the art and discussed supra. Disclosed inflammatory diseases and/or disorders are known to the art and discussed supra.

[0450] In an aspect, a subject can be in need of one or more organ transplants. In an aspect, a subj ect can have received one or more organ transplants. In an aspect, a disclosed organ transplant can comprise the heart, one or both kidneys, the liver, one or both lungs, the pancreas, part or all of the colon or the intestines, or any combination thereof.

[0451] In an aspect, a subject can be in need of gene therapy, can have received gene therapy, or can be receiving gene therapy. In an aspect, a subject can be treated with gene therapy or can have a disease and/or disorder that can be treated with gene therapy. In an aspect, a subject can have or be suspected of having a disclosed disease or disorder that can be treated with gene therapy.

[0452] In an aspect, a disclosed gene therapy can comprise the delivery and/or repair of one or more disclosed genes. In an aspect, a disclosed gene therapy can comprise AAV-based gene therapy, Adenovirus-based gene therapy, Lentiviral gene therapy, or the combination thereof. In an aspect, a disclosed gene therapy can comprise the delivery and/or repair of one or more disclosed genes. In an aspect, a disclosed gene therapy can comprise AAV-based gene therapy, Adenovirus-based gene therapy, Lentiviral gene therapy, or the combination thereof. [0453] Genetic diseases and/or disorders or diseases and/or disorders that can be treated with gene therapy are known to the art and are discussed supra. In an aspect, a disclosed gene therapy can comprise the delivery' and/or repair of one or more disclosed genes. Genetic diseases and/or disorders can be due to a defect in one or more genes known to the art and discussed supra.

[0454] In an aspect, a disclosed method of inhibiting complement activation can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme delivered by gene therapy.

[0455] In an aspect, a disclosed method of inhibiting complement activation can comprise restoring one or more aspects of cellular homeostasis and/or cellular functionality’ and/or metabolic dysregulation.

[0456] In an aspect, restoring one or more aspect of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation comprises restoring the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0457] In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell ty pes; (ii) normalizing aspects of the autophagy' pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity⁷.

[0458] In an aspect, restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme can comprise a 10%, 20%, 30%, 40%, 50%, 60%. 70%. 80%, 90%, 100%, or any amount of restoration w'hen compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.

[0459] In an aspect of a disclosed method, techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are discussed supra.

[0460] In an aspect of a disclosed method, contacting a cell can comprising methods known to the art. For example, contacting can comprise administering to a subject one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof .

[0461] In an aspect, administering can comprise intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intra-CSF, intrathecal, intraventricular, intrahepatic, hepatic intraarterial, hepatic portal vein (HPV), or in utero administration. In an aspect, a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be administered via intra-CSF administration in combination with RNAi, antisense oligonucleotides, miRNA, one or more small molecules, one or more therapeutic agents, one or more proteasome inhibitors, one or more immune modulators, and/or a gene editing system. In an aspect, a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be administered via LNP administration. In an aspect, a disclosed composition, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be concurrently and/or serially administered to a subject via multiple routes of administration. For example, in an aspect, administering a disclosed nucleic acid molecule, a disclosed enzyme or disclosed recombinant enzyme, a disclosed vector, and/or a disclosed pharmaceutical formulation can comprise intravenous administration and intra-cistem magna (ICM) administration. In an aspect, administering a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can comprise IV administration and intrathecal (ITH) administration. In an aspect, a disclosed method can employ multiple routes of administration to the subject. In an aspect, a disclosed method can employ a first route of administration that can be the same or different as a second and/or subsequent routes of administration. [0462] In an aspect, a therapeutically effective amount of a disclosed enzyme or disclosed recombinant enzyme can comprise about 0.01 mg/kg body weight to about 100 mg/kg body weight. In an aspect, a disclosed enzy me or disclosed recombinant enzyme can be therapeutically effective when the dose comprises about 0.01 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg. about 15 mg/kg, about 20 mg/kg, about 25 mg/kg. about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg body weight.

[0463] In an aspect of a disclosed method of inhibiting complement activation, a therapeutically effective amount of disclosed vector can be delivered to a subject via intravenous (IV) administration and can comprise a range of about 1 x 10¹⁰ vg/kg to about 2 x 10¹⁴ vg/kg. In an aspect, for example, a disclosed vector can be administered at a dose of about 1 x 10¹¹ vg/kg to about 8 x 10¹³ vg/kg or about 1 x 10¹² vg/kg to about 8 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 10¹³ vg/kg to about 6 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of at least about 1 x 10¹⁰ vg/kg, at least about 5 x 10¹⁰ vg/kg, at least about 1 x 10¹¹ vg/kg, at least about 5 x 10¹¹ vg/kg, at least about 1 x 10¹² vg/kg, at least about 5 x 10¹² vg/kg, at least about 1 x 10¹³ vg/kg, at least about 5 x 10¹³ vg/kg, or at least about I x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered at a dose of no more than about 1 x 10¹⁰ vg/kg, no more than about 5 x 10¹⁰ vg/kg, no more than about 1 x 10¹¹ vg/kg, no more than about 5 x 10¹¹ vg/kg, no more than about 1 x 10¹² vg/kg, no more than about 5 x 10¹² vg/kg, no more than about 1 x 10¹³ vg/kg, no more than about 5 x 10¹³, or no more than about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered to a subject at a dose of about 1 x 10¹² vg/kg. In an aspect, a disclosed vector can be administered to a subject at a dose of about 1 x 10¹¹ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x I O¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total. In an aspect, a disclosed vector can be administered in a single dose, or in multiple doses (such as 2. 3, 4, 5. 6, 7, 8. 9 or 10 doses) as needed for the desired therapeutic results.

[0464] In an aspect, a disclosed method of inhibiting complement activation can further comprise administering to the subject a therapeutically effective amount of a therapeutic agent. A therapeutic agent can be any disclosed agent that effects a desired clinical outcome. [0465] In an aspect, a disclosed method of inhibiting complement activation can further comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, the method can further comprise continuing to treat the subject. In an aspect, in the presence of adverse effects, the method can further comprise modifying the treating step. Methods of monitoring a subject’s well-being can include both subjective and objective criteria (and are discussed supra). Such methods are known to the skilled person.

[0466] In an aspect, a disclosed method of inhibiting complement activation can further comprise administering to the subject a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway. In an aspect, such an agent can comprise an enzyme for enzyme replacement therapy. In an aspect, a disclosed enzyme can replace any enzy me in a dysregulated or dysfunctional metabolic or enzymatic pathway. In an aspect, a disclosed method can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.

[0467] In an aspect, a disclosed method of inhibiting complement activation can further comprise administering one or more immune modulators. In an aspect, a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, a disclosed immune modulator can be Tacrolimus. In an aspect, a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.1 mg/kg body weight to about 0.6 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary to achieve a desired clinical effect.

[0468] In an aspect, a disclosed immune modulator can be administered orally about one hour before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered orally about one hour or a few days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before or a few days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof.

[0469] In an aspect, a disclosed method of inhibiting complement activation can further comprise administering one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib). In an aspect, a proteasome inhibitor can be an agent that acts on plasma cells (e.g.. daratumumab). In an aspect, an agent that acts on a plasma cell can be melphalan hydrochloride, melphalan, pamidronate disodium, carmustine, carfilzomib, carmustine, cyclophosphamide, daratumumab, doxorubicin hydrochloride liposome, doxorubicin hydrochloride liposome, elotuzumab, melphalan hydrochloride, panobinostat, ixazomib citrate, carfilzomib, lenalidomide, melphalan, melphalan hydrochloride, plerixafor, ixazomib citrate, pamidronate disodium, panobinostat, plerixafor, pomalidomide, pomalidomide, lenalidomide, selinexor, thalidomide, thalidomide, bortezomib, selinexor, zoledronic acid, or zoledronic acid.

[0470] In an aspect, a disclosed method of inhibiting complement activation can further comprise administering one or more proteasome inhibitors or agents that act on plasma cells prior to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells concurrently with administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells subsequent to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can further comprise administering one or more proteasome inhibitors more than 1 time. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors repeatedly over time.

[0471 ] In an aspect, a disclosed method of inhibiting complement activation can further comprise administering one or more immunosuppressive agents. In an aspect, an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, anti -thymocyte globulin (ATG). cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or a combination thereof. In an aspect, a disclosed method can comprise administering one or more immunosuppressive agents more than 1 time. In an aspect, a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time. In an aspect, a disclosed method can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.

[0472] In an aspect, a disclosed method of inhibiting complement activation can further comprise administering a compound that exerts a therapeutic effect against B cells and/or a compound that targets or alters antigen presentation or humoral or cell mediated immune response. In an aspect, a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti-IGFIR antibody, a CD19 antibody (e.g., inebilizumab), an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof. Also disclosed herein are Treg infusions that can be administered as a way to help with immune tolerance (e.g., antigen specific Treg cells to AAV). [0473] In an aspect, a disclosed method can further comprise administering lipid nanoparticles (LNPs). In an aspect, LNPs can be organ-targeted. In an aspect, LNPs can be liver-targeted or testes-targeted. For example, in an aspect, mRNA therapy with LNP encapsulation for systemic delivery to a subject has the potential to restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0474] In an aspect, a disclosed method of inhibiting complement activation can further comprise plasmapheresis and immunosuppression. In an aspect, a disclosed method can comprise using immunosuppression to decrease the T cell, B cell, and /or plasma cell population, decrease the innate immune response, inflammatory response, and antibody levels in general.

[0475] In an aspect, a disclosed method can comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells and/or a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response.

[0476] In an aspect, a disclosed method can comprise modifying one or more of the disclosed steps. For example, modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject, or by changing the duration of time one or more disclosed enzymes or recombinant enzy mes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered to a subject.

[0477] In an aspect, a disclosed method can be altered by changing the amount of one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.

[0478] In as aspect, a disclosed method can comprise concurrent administration of one or more of the following: one or more disclosed enzy mes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, one or more disclosed therapeutic agents, one or more disclosed immune modulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressive agents, one or more disclosed compounds that exert therapeutic effect against B cells, one or more disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response, or any combination thereof.

[0479] In an aspect, a disclosed immune modulator can be administered prior to or after the administration of a disclosed therapeutic agent.

[0480] In an aspect, a disclosed method of inhibiting complement activation can further comprise generating one or more disclosed enzy mes or disclosed recombinant enzy mes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed In an aspect, a disclosed method of inhibiting complement activation can further comprise validating one or more disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0481] For example, in an aspect, a disclosed method of inhibiting complement activation can further comprise generating a disclosed viral or non-viral vector. In an aspect, generating a disclosed viral vector can comprise generating an AAV vector or a recombinant AAV (such as those disclosed herein). [0482] In an aspect, a disclosed method of inhibiting complement activation can further comprise gene editing one or more relevant genes (such as, for example, a missing, deficient, and/or mutant protein or enzyme), wherein editing includes but is not limited to single gene knockout, loss of function screening of multiple genes at one, gene knockin, or a combination thereof.

[0483] In an aspect, a disclosed method of inhibiting complement activation can further comprise administering an oligonucleotide therapeutic agent. A disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, noncoding RNA (ncRNA), an antisense molecule, miRNA. a morpholino, a peptide-nucleic acid (PNA). or an analog or conjugate thereof. In an aspect, a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise a CRISPR-based endonuclease. In an aspect, a disclosed endonuclease can be Cas9. In an aspect, a disclosed Cas9 can be from Staphylococcus aureus or Streptococcus pyogenes.

[0484] In an aspect of a disclosed method, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered one or more times. In an aspect, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered for the duration of the subject’s life, or for the duration of the subject's treatment with, for example, gene therapy, enzyme replacement therapy, protein replacement therapy, and/or recombinant product. In an aspect, a disclosed enz me, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered every' 6 weeks, every' 2 months, every' 3 months, every' 6 months, every year, year other year, every 3 years, every 5 years, or for any other period deemed necessary by a skilled clinician.

[0485] In an aspect of a disclosed method, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered prior to, concurrent, or after the delivery and/or administration of enzyme replacement therapy, protein replacement, gene therapy, a recombinant product, or any combination thereof.

[0486] In an aspect, a disclosed method of inhibiting complement activation can further comprise reducing and/or minimizing vector-mediated immunotoxicity' and/or transgene immunogenicity' (e.g., the ability to induce specific immunity). In an aspect, vector-mediated immunotoxicity’ and/or transgene immunogenicity can decrease and/or reduce the efficacy of the recombinant product encoded by the transgene. In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subject with one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subject with gene therapy, enzyme replacement therapy, protein replacement, or any combination thereof.

[0487] In an aspect of a disclosed method of inhibiting complement activation, IgM can be cleaved in the heavy chain at the interdomain region between constant domains Cp2 and Cp3. In an aspect, IgM can be cleaved between position 350 and position 365 of SEQ ID NO: 105. In an aspect, SEQ ID NO: 105 can represent the linear sequence of an IgM (for example, the substrate). In an aspect, IgM can be cleaved between positions 355 through 360 of IgM. In an aspect, IgM can be cleaved at SEQ ID NO:110. In an aspect, IgM can be cleaved at the sequence comprising 355D, 356T, 357A, 3581, 359R, and 360V. In an aspect, IgM can be cleaved at position 355 through position 360 of the IgM of SEQ ID NO: 105. In an aspect, IgG can be cleaved below the hinge region, thereby yielding F(ab')z and Fc fragments. In an aspect. IgG can be cleaved to separate the Cp2 and Cp3 domains from the Cpl domain. In an aspect, IgG can be cleaved at SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO:113, SEQ ID NO:114, or any combination thereof. In an aspect, IgG can be cleaved at SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof. In an aspect, IgG can be cleaved at positions 109 through 124 of the IgG of SEQ ID NO: 106, positions 106 through 120 of the IgG of SEQ ID NO: 107, positions 156 through 171 of the IgG of SEQ ID NO: 108, positions 106 through 121 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, IgG can be cleaved at positions 116 through 122 of the IgG of SEQ ID NO: 106, positions 112 through 118 of the IgG of SEQ ID NO: 107, positions 163 through 169 of the IgG of SEQ ID NO: 108. positions 113 through 119 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, IgG can be cleaved SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO:118, or any combination thereof.

[0488] Disclosed herein is method of inhibiting complement activation the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes having IgM specific protease activity and one or more disclosed enzymes having IgG specific protease activity⁷.

[0489] Disclosed herein is method of inhibiting complement activation the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes having IgM specific protease activity and one or more disclosed recombinant enzymes comprising the sequence of any one SEQ ID NO: 119 - SEQ ID NO: 159. [0490] Disclosed herein is method of inhibiting complement activation the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46 and one or more disclosed recombinant enzymes comprising the sequence of any one SEQ ID NO: 119 - SEQ ID NO: 159.

[0491] Disclosed herein is method of inhibiting complement activation the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes having IgM specific protease activity and one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 119.

[0492] Disclosed herein is method of inhibiting complement activation the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes having IgM specific protease activity and one or more disclosed recombinant enzymes comprising the sequence of any one SEQ ID NO: 1 19 - SEQ ID NO: 159. [0493] Disclosed herein is method of inhibiting complement activation the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 11 or SEQ ID NO: 12 and one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 119.

[0494] Disclosed herein is method of inhibiting complement activation the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 14 or SEQ ID NO: 15 and one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 119.

[0495] In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of SEQ ID NO: 119. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 1 19. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identify to the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identify to the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159.

[0496] In an aspect, a disclosed method can further comprise administering one or more times one or more disclosed enzymes or recombinant enzy mes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0497] In an aspect, a disclosed method can further comprise measuring and/or determining a subject’s pre-treatment level of circulating IgG and/or IgM and/or a subject’s pre-treatment level of surface bound IgM. In an aspect, a disclosed method can further comprise measuring and/or determining one or more times a subject’s level of circulating IgG and/or IgM and/or a subject's level of surface bound IgM (e.g., before, during, and after a disclosed administering step).

[0498] In an aspect, a disclosed method can be performed using a singular disclosed enzyme having both IgG and IgM specific protease activity. In an aspect, a disclosed method can be performed using a disclosed enzy me having IgG specific protease activity and a disclosed enzyme having IgM specific protease activity.

G. Methods of Reducing and/or Minimizing an Immune Response

[0499] Disclosed herein is a method of reducing and/or minimizing an immune response in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed enzymes or disclosed recombinant enzymes, one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any' combination thereof.

[0500] Disclosed herein is a method of reducing and/or minimizing an immune response in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO: 15 or SEQ ID NO:24 - SEQ ID NO:38. Disclosed herein is a method of reducing and/or minimizing an immune response in a subject, the method comprising administering to the subj ect in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO:02, SEQ ID NO:03, SEQ ID NO:05, SEQ ID NO:06, SEQ ID NO:08, SEQ ID NO:09, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 15.

[0501] Disclosed herein is a method of reducing and/or minimizing an immune response in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO:47 - SEQ ID N0:61 or SEQ ID NO:70 - SEQ ID NO:84. Disclosed herein is a method of reducing and/or minimizing an immune response in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55. SEQ ID NO:57, SEQ ID NO:58. SEQ ID NO:60, or SEQ ID NO:61. Disclosed herein is a method of reducing and/or minimizing an immune response in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:74. SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, or SEQ ID NO:84.

[0502] Disclosed herein is a method of reducing and/or minimizing an immune response in a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG specific protease activity, and (ii) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgM specific protease activity.

[0503] Disclosed herein is a method of reducing and/or minimizing an immune response in a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG specific protease activity (such as, for example, the enzyme of SEQ ID NO:08, SEQ ID NO:09, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 14, or SEQ ID NO: 15), and (ii) a therapeutically effective amount of one or more disclosed recombinant enz mes having IgM specific protease activity (such as, for example, the enzyme of any one of SEQ ID NO: 119 - SEQ ID NO: 159).

[0504] Disclosed herein is a method of reducing and/or minimizing an immune response in a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG specific protease activity (such as, for example, the enzyme of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 15). and (ii) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgM specific protease activity (such as, for example, the enzyme of SEQ ID NO: 119).

[0505] Disclosed herein is a method of reducing and/or minimizing an immune response in a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO:47 - SEQ ID NO:61 and (ii) a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. [0506] Disclosed herein is a method of reducing and/or minimizing an immune response in a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of the recombinant enzyme of SEQ ID NO:08, SEQ ID NO:09, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14. or SEQ ID NO: 15, and (ii) a therapeutically effective amount of the recombinant enzyme of SEQ ID NO: 119, SEQ ID NO: 120, or SEQ ID NO: 121.

[0507] Disclosed herein is a method of reducing and/or minimizing an immune response in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161.

[0508] Disclosed herein is a method of reducing and/or minimizing an immune response in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzy me comprising the sequence set forth in any one of SEQ ID NO: 16 - SEQ ID NO:23 or SEQ ID NO:39 - SEQ ID NO:46. Disclosed herein is method of reducing and/or minimizing an immune response in a subject, the method comprising administenng to the subj ect in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO: 23.

[0509] Disclosed herein is a method of reducing and/or minimizing an immune response, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed recombinant enzy mes having IgM specific protease activity, wherein following the administering step, the level of circulating IgM targeting a recombinant product is reduced when compared to a pre-administering step level.

[0510] Disclosed herein is a method of reducing and/or minimizing an immune response, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG specific protease activity⁷, wherein following the administering step, the level of circulating IgG targeting a recombinant product is reduced when compared to a pre-administering step level.

[051 1 ] Disclosed herein is a method of reducing and/or minimizing an immune response, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed recombinant enzy mes having IgM specific protease activity⁷ and IgG specific protease activity, wherein following the administering step, the level of circulating IgM and IgG targeting a recombinant product is reduced when compared to a pre-administering step level.

[0512] Disclosed herein is a method of reducing and/or minimizing an immune response, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG specific protease activity, one or more disclosed recombinant enzymes having IgM specific protease activity, one or more disclosed recombinant enzy me having both IgM and IgG specific protease activity, or any combination thereof wherein following the administering step, the level of circulating IgG and/or IgM targeting a recombinant product is reduced when compared to a pre-administering step level.

[0513] In an aspect, a disclosed method can further comprise re-dosing the subject with the recombinant product. In an aspect, a disclosed recombinant product can comprise a vector or portion thereof. In an aspect, a disclosed recombinant product can comprise an encoded therapeutic protein or enzyme or an encoded gene product. In an aspect, a disclosed recombinant product can comprise a therapeutic protein or a therapeutic enzyme. In an aspect, a disclosed method can further comprise reducing and/or minimizes immunogenicity⁷ in the subject to the recombinant product. In an aspect, a disclosed method can further comprise improving and/or enhancing transgene efficacy and/or expression in a subject. In an aspect of a disclosed method, transgene efficacy and/or expression in the subject can be improved and/or enhanced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% when compared to a pre-administering step level.

[0514] In an aspect of a disclosed method of reducing and/or minimizing an immune response, the formation of the Cl complex and/or C3 complex can be blocked and/or minimized. In an aspect of a disclosed method of reducing and/or minimizing an immune response, the activation of the classical complement pathway can be blocked and/or minimized. In an aspect, a disclosed method of reducing and/or minimizing an immune response can transiently block and/or minimize activation of the formation of the Cl complex and/or C3 complex, can transiently block and/or minimize activation of the classical complement pathway, or any combination thereof.

[0 15] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise clearing and/or removing IgM and/or IgG and IgM from circulation in a subject, can further comprise cleaving and/or degrading circulating IgM and/or IgG and IgM in a subject, can further comprise cleaving and/or degrading all or nearly all circulating IgM and/or IgG and IgM in a subject, or any combination thereof.

[0516] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise transiently clearing and/or removing IgM and/or IgG and IgM from circulation in a subject, or transiently clearing and/or degrading circulating IgM and/or IgG and IgM in a subject.

[0517] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise completely or nearly completely clearing and/or removing IgM and/or IgG and IgM from circulation in a subject. In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise reducing and/or diminishing the level of circulating IgM and/or IgG and IgM in a subject, or transiently reducing and/or diminishing the level of circulating IgM and/or IgG and IgM in a subject.

[0518] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise clearing and/or removing IgM and/or IgG and IgM from circulation in a subject, completely or nearly completely clearing and/or removing IgM and/or IgG and IgM from circulation in a subject, clearing and/or degrade circulating IgM and/or IgG and IgM in a subject, cleaving and/or degrading all or nearly all circulating IgM and/or IgG and IgM in a subject, or any combination thereof.

[0519] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise transiently clearing and/or removing IgM and/or IgG and IgM from circulation in a subject, or can transiently cleaving and/or degrading circulating IgM and/or IgG and IgM in a subject. In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise reducing and/or diminishing the level of circulating IgM and/or IgG and IgM in a subject, or can transiently reduce and/or diminish the level of circulating IgM and/or IgG and IgM in a subject. In aspect, a disclosed transient reduction and/or diminishment can comprise about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[0520] In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against an exogenously administered protein. In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against enzyme replacement therapy or protein replacement therapy. In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against a non-viral vector or a viral vector. In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against a protein associated with a non-viral vector or a viral vector. For example, in an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against a viral vector capsid protein (e.g., an AAV capsid protein). In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against any disclosed recombinant product..

[0521] In an aspect, a disclosed method can further comprise diagnosing a subject as being in need of one or more disclosed enzymes, one or more disclosed recombinant enzymes, one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0522] In an aspect of a disclosed method, there is at least a 10% reduction, at least a 20% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, at least an 80% reduction, at least a 90% reduction, or at least a 99% reduction in the level of circulating intact IgM and/or intact IgG.

[0523] In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against one or more transplanted organs. In an aspect, a disclosed method can degrade one or more anti-HLA antibodies directed at the one or more transplanted organs.

[0524] In an aspect, a subject can have a high sustained antibody titer (HSAT - defined as titers of > 12,800). In an aspect, a subject can have a high sustained antibody titer (HSAT) against one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more non-viral vectors or viral vectors, one or more proteins associated with a non-viral vector, one or more proteins associated with a viral vector, or any combination thereof. In an aspect, after the contacting step, a disclosed HSAT can be reduced and/or eliminated. In an aspect, after the reduction and/or elimination of one or more HSATs, a subject can be re-dosed. In an aspect, after the reduction and/or elimination of one or more HSATs, a subject can be re-dosed with one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more recombinant products, one or more non-viral vectors or viral vectors, one or more proteins associated with a non-viral vector, one or more proteins associated with a viral vector, or any combination thereof.

[0525] In an aspect, a disclosed method can further comprise measuring one or more time the levels of IgM and/or IgG in the subject (such as, for example, in the subject’s sera).

[0526] In an aspect, a disclosed method of reducing and/or minimizing an immune response can enable and/or allow vector re-dosing in a subject. In an aspect, a disclosed method of reducing and/or minimizing an immune response can enable and/or allow re-dosing of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof in a subj ect.

[0527] In an aspect, a disclosed method of reducing and/or minimizing an immune response can reduce and/or minimize transgene immunogenicity in a subject, can improve and/or enhance transgene efficacy and/or expression in a subject, or the combination thereof.

[0528] In an aspect, improving and/or enhancing transgene efficacy and/or expression in a subject can comprise a 10%. 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of an improvement and/or enhancement when compared to a pre-existing level of transgene efficacy and/or expression such as, for example, a pre-treatment level (prior to administering one or more disclosed enzy mes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, the amount of improvement and/or enhancement can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as a pre-treatment level (prior to administering one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, an improvement and/or enhancement can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having received or not having been administered one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, an improvement and/or enhancement can be a partial or incomplete restoration. In an aspect, an improvement and/or enhancement can be complete or near complete such that the level of neutralizing antibodies or neutralizing IgG and/or IgM molecules is similar to that of a wildtype or control level.

[0529] In an aspect, a disclosed method of reducing and/or minimizing an immune response can remove and/or cleave surface-bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells, can transiently remove and/or cleave surface-bound IgM from B cells and/or can transiently remove and/or cleave IgM from the surface of B cells, or the combination thereof. In an aspect, the disclosed removal and/or cleavage of IgM from the surface of B cells can be reversible and/or transient. In an aspect, a disclosed method can remove and/or cleave surface bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells without killing and/or injuring the B cells.

[0530] In an aspect, a disclosed method of reducing and/or minimizing an immune response can modulate humoral immunity in a subject. In an aspect, modulating humoral immunity in a subject can be an increase in the functionality of the subject’s humoral immunity, or can be a decrease in the functional of the subject's humoral immunity.

[0531] In an aspect of a disclosed method, intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an administered gene therapy. In an aspect of a disclosed method, intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an administered therapeutic protein or a therapeutic recombinant protein. In an aspect of a disclosed method, the intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an encoded therapeutic protein or encoded therapeutic recombinant protein.

[0532] In an aspect, a disclosed method of reducing and/or minimizing an immune response can comprise administering to a subject in need thereof a theoretically effectively amount of one or more disclosed enzymes or disclosed recombinant enzymes, one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0533] In an aspect, a subject can have or can be suspected of having one of more autoimmune diseases and/or disorders, one or more autoinfl ammatory diseases and/or disorders, one or more inflammatory diseases and/or disorders, or any combination thereof.

[0534] Disclosed autoimmune diseases and/or disorders are known to the art and discussed supra. Disclosed autoinflammatory diseases and/or disorders are know n to the art and discussed supra. Disclosed inflammatory diseases and/or disorders are known to the art and discussed supra.

[0535] In an aspect, a subject can be in need of one or more organ transplants. In an aspect, a subj ect can have received one or more organ transplants. In an aspect, a disclosed organ transplant can comprise the heart, one or both kidneys, the liver, one or both lungs, the pancreas, part or all of the colon or the intestines, or any combination thereof. In an aspect, a subject can be in need of gene therapy, can have received gene therapy, or can be receiving gene therapy. In an aspect, a subject can be treated with gene therapy or can have a disease and/or disorder that can be treated with gene therapy. In an aspect, a subject can have or be suspected of having a disclosed disease or disorder that can be treated with gene therapy.

[0536] In an aspect, a disclosed gene therapy can comprise the delivery and/or repair of one or more disclosed genes. In an aspect, a disclosed gene therapy can comprise AAV-based gene therapy, Adenovirus-based gene therapy, Lentiviral gene therapy, or the combination thereof. In an aspect, a disclosed gene therapy can comprise the delivery and/or repair of one or more disclosed genes. In an aspect, a disclosed gene therapy can comprise AAV-based gene therapy, Adenovirus-based gene therapy, Lentiviral gene therapy, or the combination thereof.

[0537] Genetic diseases and/or disorders or diseases and/or disorders that can be treated with gene therapy are known to the art and are discussed supra. In an aspect, a disclosed gene therapy can comprise the delivery and/or repair of one or more disclosed genes. Genetic diseases and/or disorders can be due to a defect in one or more genes known to the art and discussed supra.

[0538] In an aspect, a disclosed method of reducing and/or minimizing an immune response can restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme delivered by gene therapy.

[0539] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise restoring one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation. In an aspect, restoring one or more aspect of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation comprises restoring the functionality' and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme. [0540] In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy' pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (iii) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi-systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.

[0541] In an aspect, restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%. 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.

[0542] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise administering to the subject a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway. In an aspect, such an agent can comprise an enzyme for enzyme replacement therapy. In an aspect, a disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway. In an aspect, a disclosed method can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.

[0543] In an aspect of a disclosed method, techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are discussed supra. [0544] In an aspect of a disclosed method, contacting a cell can comprising methods know n to the art. For example, contacting can comprise administering to a subject one or more disclosed enzy mes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof .

[0545] In an aspect, administering can comprise intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intra-CSF, intrathecal, intraventricular, intrahepatic, hepatic intraarterial, hepatic portal vein (HPV), or in utero administration. In an aspect, a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be administered via intra-CSF administration in combination with RNAi, antisense oligonucleotides, miRNA, one or more small molecules, one or more therapeutic agents, one or more proteasome inhibitors, one or more immune modulators, and/or a gene editing system. In an aspect, a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be administered via LNP administration. In an aspect, a disclosed composition, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be concurrently and/or serially administered to a subject via multiple routes of administration. For example, in an aspect, administering a disclosed nucleic acid molecule, a disclosed enzyme or disclosed recombinant enzyme, a disclosed vector, and/or a disclosed pharmaceutical formulation can comprise intravenous administration and intra-cistem magna (ICM) administration. In an aspect, administering a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can comprise IV administration and intrathecal (ITH) administration. In an aspect, a disclosed method can employ multiple routes of administration to the subject. In an aspect, a disclosed method can employ a first route of administration that can be the same or different as a second and/or subsequent routes of administration.

[0546] In an aspect, a therapeutically effective amount of a disclosed enzyme or disclosed recombinant enzyme can comprise about 0.01 mg/kg body weight to about 100 mg/kg body weight. In an aspect, a disclosed enzyme or disclosed recombinant enzyme can be therapeutically effective when the dose comprises about 0.01 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg body weight. [0547] In an aspect of a disclosed method of reducing and/or minimizing an immune response, a therapeutically effective amount of disclosed vector can be delivered to a subject via intravenous (IV) administration and can comprise a range of about 1 x IO¹⁰ vg/kg to about 2 x 10¹⁴ vg/kg. In an aspect, for example, a disclosed vector can be administered at a dose of about 1 x 10¹¹ vg/kg to about 8 x 10¹³ vg/kg or about I x 10¹² vg/kg to about 8 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 10¹³ vg/kg to about 6 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of at least about 1 x IO¹⁰ vg/kg, at least about 5 x IO¹⁰ vg/kg, at least about 1 x 10¹¹ vg/kg, at least about 5 x 10¹¹ vg/kg, at least about 1 x 10¹² vg/kg, at least about 5 x 10¹² vg/kg. at least about 1 x 10¹³ vg/kg. at least about 5 x 10¹³ vg/kg. or at least about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered at a dose of no more than about 1 x IO¹⁰ vg/kg, no more than about 5 x IO¹⁰ vg/kg, no more than about 1 x 10¹¹ vg/kg, no more than about 5 x 10¹¹ vg/kg, no more than about 1 x 10¹² vg/kg, no more than about 5 x 10¹² vg/kg, no more than about 1 x 10¹³ vg/kg, no more than about 5 x 10¹³, or no more than about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered to a subject at a dose of about 1 x 10¹² vg/kg. In an aspect, a disclosed vector can be administered to a subject at a dose of about 1 x 10¹¹ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total. In an aspect, a disclosed vector can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.

[0548] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise administering to the subject a therapeutically effective amount of a therapeutic agent. A therapeutic agent can be any disclosed agent that effects a desired clinical outcome.

[0549] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, the method can further comprise continuing to treat the subject. In an aspect, in the presence of adverse effects, the method can further comprise modifying the treating step. Methods of monitoring a subject’s well-being can include both subjective and objective criteria (and are discussed supra). Such methods are known to the skilled person.

[0550] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise administering one or more immune modulators. In an aspect, a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In an aspect, a disclosed immune modulator can be bortezomib or SVP- Rapamycin. In an aspect, a disclosed immune modulator can be Tacrolimus. In an aspect, a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose. In an aspect, a disclosed immune modulator can be administered at a dose of about 0. 1 mg/kg body weight to about 0.6 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary’ to achieve a desired clinical effect.

[0551] In an aspect, a disclosed immune modulator can be administered orally about one hour before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered orally about one hour or a few days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before or a few days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof.

[0552] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise administering one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib). In an aspect, a proteasome inhibitor can be an agent that acts on plasma cells (e.g., daratumumab). In an aspect, an agent that acts on a plasma cell can be melphalan hydrochloride, melphalan, pamidronate disodium, carmustine, carfilzomib, carmustine, cyclophosphamide, daratumumab, doxorubicin hydrochloride liposome, doxorubicin hydrochloride liposome, elotuzumab, melphalan hydrochloride, panobinostat, ixazomib citrate, carfilzomib, lenalidomide, melphalan, melphalan hydrochloride, plerixafor, ixazomib citrate, pamidronate disodium, panobinostat, plerixafor, pomalidomide, pomalidomide, lenalidomide, selinexor, thalidomide, thalidomide, bortezomib, selinexor, zoledronic acid, or zoledronic acid. [0553] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise administering one or more proteasome inhibitors or agents that act on plasma cells prior to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells concurrently with administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells subsequent to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can further comprise administering one or more proteasome inhibitors more than 1 time. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors repeatedly over time.

[0554] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise administering one or more immunosuppressive agents. In an aspect, an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, antithymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or a combination thereof. In an aspect, a disclosed method can comprise administering one or more immunosuppressive agents more than 1 time. In an aspect, a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time. In an aspect, a disclosed method can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.

[0555] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further compnse administering a compound that exerts a therapeutic effect against B cells and/or a compound that targets or alters antigen presentation or humoral or cell mediated immune response. In an aspect, a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti-IGFIR antibody, a CD19 antibody (e.g., inebilizumab), an anti-IL6 antibody (e.g.. tocihzumab), an antibody to CD40, an IL2 mutein, or a combination thereof. Also disclosed herein are Treg infusions that can be administered as a way to help with immune tolerance (e.g., antigen specific Treg cells to AAV).

[0556] In an aspect, a disclosed method can further comprise administering lipid nanoparticles (LNPs). In an aspect, LNPs can be organ-targeted. In an aspect, LNPs can be liver-targeted or testes-targeted. For example, in an aspect, mRNA therapy with LNP encapsulation for systemic delivery⁷ to a subject has the potential to restore the functionality and/or structural integrity' of a missing, deficient, and/or mutant protein or enzyme. [0557] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise plasmapheresis and immunosuppression. In an aspect, a disclosed method can comprise using immunosuppression to decrease the T cell, B cell, and /or plasma cell population, decrease the innate immune response, inflammatory response, and antibody levels in general.

[0558] In an aspect, a disclosed method can comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells and/or a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response.

[0559] In an aspect, a disclosed method can comprise modifying one or more of the disclosed steps. For example, modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject, or by changing the duration of time one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered to a subject.

[0560] In an aspect, a disclosed method can be altered by changing the amount of one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.

[0561] In as aspect, a disclosed method can comprise concurrent administration of one or more of the following: one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, one or more disclosed therapeutic agents, one or more disclosed immune modulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressive agents, one or more disclosed compounds that exert therapeutic effect against B cells, one or more disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response, or any combination thereof.

[0562] In an aspect, a disclosed immune modulator can be administered prior to or after the administration of a disclosed therapeutic agent.

[0563] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise generating and/or validating one or more disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. [0564] For example, in an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise generating a disclosed viral or non-viral vector. In an aspect, generating a disclosed viral vector can comprise generating an AAV vector or a recombinant AAV (such as those disclosed herein).

[0565] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise gene editing one or more relevant genes (such as. for example, a missing, deficient, and/or mutant protein or enzyme), wherein editing includes but is not limited to single gene knockout, loss of function screening of multiple genes at one, gene knockin, or a combination thereof.

[0566] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise administering an oligonucleotide therapeutic agent. A disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof. In an aspect, a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise a CRISPR- based endonuclease. In an aspect, a disclosed endonuclease can be Cas9. In an aspect, a disclosed Cas9 can be from Staphylococcus aureus or Streptococcus pyogenes.

[0567] In an aspect of a disclosed method, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered one or more times. In an aspect, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered for the duration of the subject’s life, or for the duration of the subject's treatment with, for example, gene therapy, enzy me replacement therapy, protein replacement therapy, and/or recombinant product. In an aspect, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered every' 6 weeks, every' 2 months, every' 3 months, every' 6 months, every' year, year other year, every 3 years, every 5 years, or for any other period deemed necessary by a skilled clinician.

[0568] In an aspect of a disclosed method, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered prior to, concurrent, or after the delivery and/or administration of enzyme replacement therapy, protein replacement, gene therapy, recombinant product, or any combination thereof.

[0569] In an aspect, a disclosed method of reducing and/or minimizing an immune response can further comprise reducing and/or minimizing vector-mediated immunotoxicity' and/or transgene immunogenicity (e.g., the ability to induce specific immunity). In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the efficacy of the recombinant product encoded by the transgene. In an aspect, vector-mediated immunotoxicity' and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of redosing a subject with one or more disclosed enzy mes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subject with gene therapy, enzyme replacement therapy, protein replacement, or any combination thereof.

[0570] In an aspect of a disclosed method of reducing and/or minimizing an immune response, IgM can be cleaved in the heavy chain at the interdomain region between constant domains Cp2 and Cp3. In an aspect, IgM can be cleaved between position 350 and position 365 of SEQ ID NO: 105. In an aspect, SEQ ID NO: 105 can represent the linear sequence of an IgM (for example, the substrate). In an aspect, IgM can be cleaved between positions 355 through 360 of IgM. In an aspect, IgM can be cleaved at SEQ ID NO: 110. In an aspect, IgM can be cleaved at the sequence comprising 355D, 356T, 357A, 3581, 359R, and 360V. In an aspect, IgM can be cleaved at position 355 through position 360 of the IgM of SEQ ID NO: 105. In an aspect, IgG can be cleaved below the hinge region, thereby yielding F(ab’)z and Fc fragments. In an aspect, IgG can be cleaved to separate the Cp2 and Cp3 domains from the Cpl domain. In an aspect, IgG can be cleaved at SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or any combination thereof. In an aspect, IgG can be cleaved at SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof. In an aspect, IgG can be cleaved at positions 109 through 124 of the IgG of SEQ ID NO: 106, positions 106 through 120 of the IgG of SEQ ID NO: 107, positions 156 through 171 of the IgG of SEQ ID NO: 108, positions 106 through 121 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, IgG can be cleaved at positions 116 through 122 of the IgG of SEQ ID NO: 106, positions 112 through 118 of the IgG of SEQ ID NO: 107. positions 163 through 169 of the IgG of SEQ ID NO: 108, positions 113 through 119 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, IgG can be cleaved SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof.

[0571] In an aspect, a disclosed method can further comprise administering one or more times one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0572] In an aspect, a disclosed method can further comprise measuring and/or determining a subject's pre-treatment level of circulating IgG and/or IgM and/or a subject’s pre-treatment level of surface bound IgM. In an aspect, a disclosed method can further comprise measuring and/or determining one or more times a subject’s level of circulating IgG and/or IgM and/or a subject’s level of surface bound IgM (e.g., before, during, and after a disclosed administering step).

[0573] In an aspect, a disclosed method can be performed using a singular disclosed enzyme having both IgG and IgM specific protease activity. In an aspect, a disclosed method can be performed using a disclosed enzyme having IgG specific protease activity and a disclosed enzyme having IgM specific protease activity.

H. Methods of Improving and Enhancing Transgene Efficacy and/or Expression

[0574] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed enzymes or disclosed recombinant enzymes. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed nucleic acid molecules. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed vectors. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed pharmaceutical formulations.

[0575] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO: 15. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:38. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO:02 or SEQ ID NO:03. Disclosed herein is method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO:05 or SEQ ID NO:06. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO: 08 or SEQ ID NO:09. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15.

[0576] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO:47 - SEQ ID NO:61 or SEQ ID NO:70 - SEQ ID NO:84. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, or SEQ ID NO:61. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:78. SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, or SEQ ID NO: 84.

[0577] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed enzy mes or disclosed recombinant enzymes. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed nucleic acid molecules. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of one or more disclosed vectors. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subj ect in need thereof a therapeutically effective amount of one or more disclosed pharmaceutical formulations.

[0578] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzy me comprising the sequence set forth in any one of SEQ ID NO: 16 - SEQ ID NO:23. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in any one of SEQ ID NO:39 - SEQ ID NO:46. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression on in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of an enzyme comprising the sequence set forth in SEQ ID NO: 22 or SEQ ID NO:23. [0579] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO: 62 - SEQ ID NO: 69 or SEQ ID NO: 85 - SEQ ID NO: 92. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO 67, SEQ ID NO:68, or SEQ ID NO:69. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 86, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, or SEQ ID NO:92.

[0580] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG specific protease activity, and (ii) a therapeutically effective amount of one or more disclosed recombinant enzy mes having IgM specific protease activity.

[0581] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG specific protease activity (such as, for example, the enzyme of SEQ ID NO:08, SEQ ID NO:09, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 15). and (ii) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgM specific protease activity’ (such as, for example, the enzyme of any one of SEQ ID NO: 119 - SEQ ID NO: 159).

[0582] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgG specific protease activity’ (such as, for example, the enzyme of SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 15), and (ii) a therapeutically effective amount of one or more disclosed recombinant enzymes having IgM specific protease activity (such as, for example, the enzyme of SEQ ID NO: 119).

[0583] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof (i) a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO:47 - SEQ ID N0:61 and (ii) a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161.

[0584] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, method comprising administering to the subject in need thereof (i) a therapeutically effective amount of the recombinant enzyme of SEQ ID NO:08, SEQ ID NO:09, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 15, and (ii) a therapeutically effective amount of the recombinant enzy me of SEQ ID NO: 119, SEQ ID NO: 120, or SEQ ID NO: 121.

[0585] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO 161.

[0586] In an aspect of a disclosed method of improving and/or enhancing transgene efficacy and/or expression, the formation of the Cl complex and/or C3 complex can be blocked and/or minimized. In an aspect of a disclosed method of improving and/or enhancing transgene efficacy and/or expression, the activation of the classical complement pathway can be blocked and/or minimized. In an aspect, a disclosed method can transiently block and/or minimize activation of the complement cascade, can transiently block and/or minimize activation of the formation of the Cl complex and/or C3 complex, can transiently block and/or minimize activation of the classical complement pathway, or any combination thereof.

[0587] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise clearing and/or removing IgM and/or IgG and IgM from circulation in a subject, can further comprise cleaving and/or degrading circulating IgM and/or IgG and IgM in a subject, can further comprise cleaving and/or degrading all or nearly all circulating IgM and/or IgG and IgM in a subject, or any combination thereof.

[0588] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise transiently clearing and/or removing IgM and/or IgG and IgM from circulation in a subject, or transiently clearing and/or degrading circulating IgM and/or IgG and IgM in a subject, can further comprise completely or nearly completely clearing and/or removing IgM and/or IgG and IgM from circulation in a subject, can further comprise reducing and/or diminishing the level of circulating IgM and/or IgG and IgM in a subject, or transiently reducing and/or diminishing the level of circulating IgM and/or IgG and IgM in a subject, or any combination thereof.

[0589] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise clearing and/or removing IgM and/or IgG and IgM from circulation in a subject, can further comprise clearing and/or degrade circulating IgM and/or IgG and IgM in a subject, can further comprise cleaving and/or degrading all or nearly all circulating IgM and/or IgG and IgM in a subject, can further comprise transiently clearing and/or removing IgM and/or IgG and IgM from circulation in a subject, can further comprise transiently cleaving and/or degrading circulating IgM and IgM in a subject, or any combination thereof.

[0590] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise completely or nearly completely clearing and/or removing IgM and/or IgG and IgM from circulation in a subject, can further comprise reducing and/or diminishing the level of circulating IgM and/or IgG and IgM in a subject, can further transiently reduce and/or diminish the level of circulating IgM and/or IgG and IgM in a subject, or any combination thereof. In aspect, a disclosed transient reduction and/or diminishment can comprise about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, or more than 4 weeks.

[05 1] In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against an exogenously administered protein. In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against enzyme replacement therapy or protein replacement therapy or recombinant product. In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against a non-viral vector or a viral vector. In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against a protein associated with a non-viral vector or a viral vector. For example, in an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against a viral vector capsid protein (e.g., an AAV capsid protein).

[0592] In an aspect, a disclosed method can further comprise diagnosing a subject as being in need of one or more disclosed enzy mes, one or more disclosed recombinant enzymes, one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0593] In an aspect of a disclosed method, there is at least a 10% reduction, at least a 20% reduction, at least a 30% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, at least an 80% reduction, at least a 90% reduction, or at least a 99% reduction in the level of circulating intact IgM and/or intact IgG.

[0594] In an aspect, a disclosed IgM molecule and/or a disclosed IgG molecule can be neutralizing against one or more transplanted organs. In an aspect, a disclosed method can degrade one or more anti-HLA antibodies directed at the one or more transplanted organs. [0595] In an aspect, a subject can have a high sustained antibody titer (HSAT - defined as titers of > 12,800). In an aspect, a subject can have a high sustained antibody titer (HSAT) against one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more non-viral vectors or viral vectors, one or more proteins associated with a non-viral vector, one or more proteins associated with a viral vector, or any combination thereof. In an aspect, after the contacting step, a disclosed HSAT can be reduced and/or eliminated. In an aspect, after the reduction and/or elimination of one or more HSATs, a subject can be re-dosed. In an aspect, after the reduction and/or elimination of one or more HSATs, a subject can be re-dosed with one or more exogenously administered proteins or enzymes, one or more enzyme replacement therapies, one or more protein replacement therapies, one or more recombinant products, one or more non-viral vectors or viral vectors, one or more proteins associated with a non-viral vector, one or more proteins associated with a viral vector, or any combination thereof.

[0596] In an aspect, a disclosed method can further compnse measuring one or more time the levels of IgM and/or IgG in the subject (such as, for example, in the subject’s sera).

[0597] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise reducing and/or minimizing vector mediated immunotoxicity in a subject. In an aspect, a disclosed method of inhibiting complement activation can reduce and/or minimize transgene immunogenicity in a subj ect.

[0598] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can enable and/or allow vector re-dosing in a subject.

[0599] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can enable and/or allow re-dosing of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof in a subject. In an aspect, a disclosed method of inhibiting complement activation can improve and/or enhance transgene efficacy and/or expression in a subject. In an aspect, improving and/or enhancing transgene efficacy and/or expression in a subject can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of an improvement and/or enhancement when compared to a pre-existing level of transgene efficacy and/or expression such as, for example, a pre-treatment level (prior to administering one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, the amount of improvement and/or enhancement can be 10-20%, 20-30%, 30-40%, 40- 50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as a pre-treatment level (prior to administering one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, an improvement and/or enhancement can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having received or not having been administered one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof). In an aspect, an improvement and/or enhancement can be a partial or incomplete restoration. In an aspect, an improvement and/or enhancement can be complete or near complete such that the level of neutralizing antibodies or neutralizing IgG and/or IgM molecules is similar to that of a wild-type or control level.

[0600] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can remove and/or cleave surface-bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells. In an aspect, the disclosed removal and/or cleavage of IgM from the surface of B cells can be reversible and/or transient. In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can transiently remove and/or cleave surface-bound IgM from B cells and/or can transiently remove and/or cleave IgM from the surface of B cells. In an aspect, a disclosed method can remove and/or cleave surface bound IgM from B cells and/or can remove and/or cleave IgM from the surface of B cells without killing and/or injuring the B cells.

[0601] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further modulate humoral immunity in a subject. In an aspect, modulating humoral immunity in a subject can be an increase in the functionality of the subject’s humoral immunity, or can be a decrease in the functional of the subject’s humoral immunity.

[0602] In an aspect of a disclosed method, intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an administered gene therapy. In an aspect of a disclosed method, intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an administered therapeutic protein or a therapeutic recombinant protein. In an aspect of a disclosed method, the intact IgM and/or intact IgG can decrease and/or diminish the effectiveness and/or efficacy of an encoded therapeutic protein or encoded therapeutic recombinant protein.

[0603] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise administering to a subject in need thereof a theoretically effectively amount of one or more disclosed enzy mes or disclosed recombinant enzymes, one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0604] In an aspect, a subject can have or can be suspected of having one of more autoimmune diseases and/or disorders, one or more autoinfl ammatory diseases and/or disorders, one or more inflammatory diseases and/or disorders, or any combination thereof. Disclosed autoimmune diseases and/or disorders are known to the art and discussed supra. Disclosed autoinflammatory diseases and/or disorders are known to the art and discussed supra. Disclosed inflammatory diseases and/or disorders are known to the art and discussed supra.

[0605] In an aspect, a subject can be in need of one or more organ transplants. In an aspect, a subj ect can have received one or more organ transplants. In an aspect, a disclosed organ transplant can comprise the heart, one or both kidneys, the liver, one or both lungs, the pancreas, part or all of the colon or the intestines, or any combination thereof.

[0606] In an aspect, a subject can be in need of gene therapy, can have received gene therapy, or can be receiving gene therapy. In an aspect, a subject can be treated with gene therapy or can have a disease and/or disorder that can be treated with gene therapy. In an aspect, a subject can have or be suspected of having a disclosed disease or disorder that can be treated with gene therapy.

[0607] In an aspect, a disclosed gene therapy can comprise the delivery and/or repair of one or more disclosed genes. In an aspect, a disclosed gene therapy can comprise AAV-based gene therapy, Adenovirus-based gene therapy, Lentiviral gene therapy, or the combination thereof. In an aspect, a disclosed gene therapy can comprise the delivery and/or repair of one or more disclosed genes. In an aspect, a disclosed gene therapy can comprise AAV-based gene therapy, Adenovirus-based gene therapy, Lentiviral gene therapy, or the combination thereof.

[0608] Genetic diseases and/or disorders or diseases and/or disorders that can be treated with gene therapy are known to the art and are discussed supra. In an aspect, a disclosed gene therapy can comprise the delivery and/or repair of one or more disclosed genes. Genetic diseases and/or disorders can be due to a defect in one or more genes known to the art and discussed supra.

[0609] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme delivered by gene therapy. In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise restoring one or more aspects of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation.

[0610] In an aspect, restoring one or more aspect of cellular homeostasis and/or cellular functionality and/or metabolic dysregulation comprises restoring the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme. In an aspect, restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (such as, for example, correcting, preventing, reducing, and/or ameliorating autophagy); (hi) improving, enhancing, restoring, and/or preserving mitochondrial functionality and/or structural integrity; (iv) improving, enhancing, restoring, and/or preserving organelle functionality and/or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of the multi- systemic manifestations of a genetic disease or disorder; (vii) reversing, inhibiting, preventing, stabilizing, and/or slowing the rate of progression of a genetic disease or disorder, or (viii) any combination thereof. In an aspect, restoring one or more aspects of cellular homeostasis can comprise improving, enhancing, restoring, and/or preserving one or more aspects of cellular structural and/or functional integrity.

[0611] In an aspect, restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%. 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzy me). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.

[0612] In an aspect of a disclosed method, techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are discussed supra.

[0613] In an aspect of a disclosed method, contacting a cell can comprising methods known to the art. For example, contacting can comprise administering to a subject one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof . [0614] In an aspect, administering can comprise intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intra-CSF, intrathecal, intraventricular, intrahepatic, hepatic intraarterial, hepatic portal vein (HPV), or in utero administration. In an aspect, a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be administered via intra-CSF administration in combination with RNAi, antisense oligonucleotides, miRNA, one or more small molecules, one or more therapeutic agents, one or more proteasome inhibitors, one or more immune modulators, and/or a gene editing system. In an aspect, a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be administered via LNP administration. In an aspect, a disclosed composition, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can be concurrently and/or serially administered to a subject via multiple routes of administration. For example, in an aspect, administering a disclosed nucleic acid molecule, a disclosed enzyme or disclosed recombinant enzyme, a disclosed vector, and/or a disclosed pharmaceutical formulation can comprise intravenous administration and intra-cistem magna (ICM) administration. In an aspect, administering a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed vector can comprise IV administration and intrathecal (ITH) administration. In an aspect, a disclosed method can employ multiple routes of administration to the subject. In an aspect, a disclosed method can employ a first route of administration that can be the same or different as a second and/or subsequent routes of administration.

[0615] In an aspect, a therapeutically effective amount of a disclosed enzyme or disclosed recombinant enzyme can comprise about 0.01 mg/kg body weight to about 100 mg/kg body weight. In an aspect, a disclosed enzyme or disclosed recombinant enzy me can be therapeutically effective when the dose comprises about 0.01 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg. about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg body weight.

[0616] In an aspect of a disclosed method of improving and/or enhancing transgene efficacy and/or expression, a therapeutically effective amount of disclosed vector can be delivered to a subject via intravenous (IV) administration and can comprise a range of about 1 x 10¹⁰ vg/kg to about 2 x 10¹⁴ vg/kg. In an aspect, for example, a disclosed vector can be administered at a dose of about 1 x 10¹¹ vg/kg to about 8 x 10¹³ vg/kg or about 1 x 10¹² vg/kg to about 8 x 10^lj vg/kg. In an aspect, a disclosed vector can be administered at a dose of about 1 x 10¹³ vg/kg to about 6 x 10¹³ vg/kg. In an aspect, a disclosed vector can be administered at a dose of at least about 1 x IO¹⁰ vg/kg, at least about 5 x IO¹⁰ vg/kg, at least about 1 x 10¹¹ vg/kg, at least about 5 x 10¹¹ vg/kg, at least about 1 x 10¹² vg/kg, at least about 5 x 10¹² vg/kg, at least about 1 x 10¹³ vg/kg, at least about 5 x 10¹³ vg/kg, or at least about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered at a dose of no more than about 1 x IO¹⁰ vg/kg, no more than about 5 x IO¹⁰ vg/kg, no more than about 1 x 10¹¹ vg/kg, no more than about 5 x 10¹¹ vg/kg, no more than about 1 x 10¹² vg/kg, no more than about 5 x 10¹² vg/kg, no more than about 1 x 10¹³ vg/kg, no more than about 5 x 10¹³, or no more than about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed vector can be administered to a subject at a dose of about 1 x 10¹² vg/kg. In an aspect, a disclosed vector can be administered to a subject at a dose of about 1 x 10¹¹ vg/kg. In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of disclosed vector can be delivered via intravenous (IV) administration and can comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total. In an aspect, a disclosed vector can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5. 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results.

[0617] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise administering to the subject a therapeutically effective amount of a therapeutic agent. A therapeutic agent can be any disclosed agent that effects a desired clinical outcome.

[0618] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, the method can further comprise continuing to treat the subject. In an aspect, in the presence of adverse effects, the method can further comprise modifying the treating step. Methods of monitoring a subject’s well-being can include both subjective and objective criteria (and are discussed supra). Such methods are known to the skilled person.

[0619] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise administering to the subject a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway. In an aspect, such an agent can comprise an enzyme for enzyme replacement therapy. In an aspect, a disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway. In an aspect, a disclosed method can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.

[0620] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise administering one or more immune modulators. In an aspect, a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, a disclosed immune modulator can be Tacrolimus. In an aspect, a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.1 mg/kg body weight to about 0.6 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary' to achieve a desired clinical effect.

[0621] In an aspect, a disclosed immune modulator can be administered orally about one hour before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed therapeutic agent. In an aspect, a disclosed immune modulator can be administered orally about one hour or a few days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered subcutaneously about 15 minutes before or a few⁷ days before a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof. In an aspect, a disclosed immune modulator can be administered concurrently with a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof.

[0622] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise administering one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib). In an aspect, a proteasome inhibitor can be an agent that acts on plasma cells (e.g., daratumumab). In an aspect, an agent that acts on a plasma cell can be melphalan hydrochloride, melphalan, pamidronate disodium, carmustine, carfilzomib. carmustine, cyclophosphamide, daratumumab, doxorubicin hydrochloride liposome, doxorubicin hydrochloride liposome, elotuzumab, melphalan hydrochloride, panobinostat, ixazomib citrate, carfilzomib, lenalidomide, melphalan, melphalan hydrochloride, plerixafor, ixazomib citrate, pamidronate disodium, panobinostat, plerixafor, pomalidomide, pomalidomide, lenalidomide, selinexor, thalidomide, thalidomide, bortezomib, selinexor, zoledronic acid, or zoledronic acid.

[0623] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise administering one or more proteasome inhibitors or agents that act on plasma cells prior to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells concurrently with administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors or one or more agents that act on plasma cells subsequent to administering a disclosed isolated nucleic acid molecule, a disclosed vector, or a disclosed pharmaceutical formulation. In an aspect, a disclosed method can further comprise administering one or more proteasome inhibitors more than 1 time. In an aspect, a disclosed method can comprise administering one or more proteasome inhibitors repeatedly over time.

[0624] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise administering one or more immunosuppressive agents. In an aspect, an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, anti -thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or a combination thereof. In an aspect, a disclosed method can comprise administering one or more immunosuppressive agents more than 1 time. In an aspect, a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time. In an aspect, a disclosed method can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.

[0625] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise administering a compound that exerts a therapeutic effect against B cells and/or a compound that targets or alters antigen presentation or humoral or cell mediated immune response. In an aspect, a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti -I GF 1R antibody, a CD 19 antibody (e.g., inebilizumab), an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof. Also disclosed herein are Treg infusions that can be administered as a way to help with immune tolerance (e.g., antigen specific Treg cells to AAV). [0626] In an aspect, a disclosed method can further comprise administering lipid nanoparticles (LNPs). In an aspect, LNPs can be organ-targeted. In an aspect, LNPs can be liver-targeted or testes-targeted. For example, in an aspect, mRNA therapy with LNP encapsulation for systemic delivery to a subject has the potential to restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0627] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise plasmapheresis and immunosuppression. In an aspect, a disclosed method can comprise using immunosuppression to decrease the T cell, B cell, and /or plasma cell population, decrease the innate immune response, inflammatory response, and antibody levels in general.

[0628] In an aspect, a disclosed method can comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed enzy me, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells and/or a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response.

[0629] In an aspect, a disclosed method can comprise modifying one or more of the disclosed steps. For example, modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by' changing the frequency of administration of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject, or by changing the duration of time one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered to a subject.

[0630] In an aspect, a disclosed method can be altered by changing the amount of one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.

[0631] In as aspect, a disclosed method can comprise concurrent administration of one or more of the following: one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, one or more disclosed therapeutic agents, one or more disclosed immune modulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressive agents, one or more disclosed compounds that exert therapeutic effect against B cells, one or more disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response, or any combination thereof.

[0632] In an aspect, a disclosed immune modulator can be administered prior to or after the administration of a disclosed therapeutic agent.

[0633] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise generating one or more disclosed enzymes or disclosed recombinant enzy mes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. [0634] For example, in an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise generating a disclosed viral or non-viral vector.

[0635] In an aspect, generating a disclosed viral vector can comprise generating an AAV vector or a recombinant AAV (such as those disclosed herein).

[0636] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise gene editing one or more relevant genes (such as, for example, a missing, deficient, and/or mutant protein or enzyme), wherein editing includes but is not limited to single gene knockout, loss of function screening of multiple genes at one, gene knockin, or a combination thereof.

[0637] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise administering an oligonucleotide therapeutic agent. A disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA. non-coding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof. In an aspect, a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise a CRISPR- based endonuclease. In an aspect, a disclosed endonuclease can be Cas9. In an aspect, a disclosed Cas9 can be from Staphylococcus aureus or Streptococcus pyogenes.

[0638] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise generating and/or validating one or more disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0639] In an aspect of a disclosed method, a disclosed enzyme, a disclosed recombinant enzy me, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered prior to, concurrent, or after the delivery and/or administration of enzyme replacement therapy, protein replacement, gene therapy, a recombinant product, or any combination thereof.

[0640] In an aspect, a disclosed method of improving and/or enhancing transgene efficacy and/or expression can further comprise reducing and/or minimizing vector-mediated immunotoxicity' and/or transgene immunogenicity (e g., the ability to induce specific immunity). In an aspect, vector-mediated immunotoxicity' and/or transgene immunogenicity' can decrease and/or reduce the efficacy of the recombinant product encoded by the transgene. In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subject with one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In an aspect, vector- mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability’ and/or likelihood of re-dosing a subject with gene therapy, enzyme replacement therapy, protein replacement, or any combination thereof.

[0641] In an aspect of a disclosed method of improving and/or enhancing transgene efficacy and/or expression, IgM can be cleaved in the heavy chain at the interdomain region between constant domains Cp2 and Cp3. In an aspect, IgM can be cleaved between position 350 and position 365 of SEQ ID NO: 105. In an aspect, SEQ ID NO: 105 can represent the linear sequence of an IgM (for example, the substrate). In an aspect, IgM can be cleaved between positions 355 through 360 of IgM. In an aspect, IgM can be cleaved at SEQ ID NO: 110. In an aspect, IgM can be cleaved at the sequence comprising 355D, 356T, 357A, 3581, 359R, and 360V. In an aspect, IgM can be cleaved at position 355 through position 360 of the IgM of SEQ ID NO: 105. In an aspect, IgG can be cleaved below the hinge region, thereby yielding F(ab’)2 and Fc fragments. In an aspect, IgG can be cleaved to separate the Cp2 and Cp3 domains from the Cpl domain. In an aspect, IgG can be cleaved at SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or any combination thereof. In an aspect, IgG can be cleaved at SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof. In an aspect, IgG can be cleaved at positions 109 through 124 of the IgG of SEQ ID NO: 106, positions 106 through 120 of the IgG of SEQ ID NO: 107. positions 156 through 171 of the IgG of SEQ ID NO: 108, positions 106 through 121 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, IgG can be cleaved at positions 116 through 122 of the IgG of SEQ ID NO: 106, positions 1 12 through 118 of the IgG of SEQ ID NO: 107, positions 163 through 169 of the IgG of SEQ ID NO: 108, positions 113 through 119 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, IgG can be cleaved SEQ ID NO: 115. SEQ ID NO: 116. SEQ ID NO: 117, SEQ ID NO: 118, or any combination thereof.

[0642] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes having IgM specific protease activity and one or more disclosed enzymes having IgG specific protease activity.

[0643] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes having IgM specific protease activity and one or more disclosed recombinant enzymes comprising the sequence of any one SEQ ID NO: 119 - SEQ ID NO: 159. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzy mes comprising the sequence of any one of SEQ ID NO:01 - SEQ ID NO:46 and one or more disclosed recombinant enzymes comprising the sequence of any one SEQ ID NO: 119 - SEQ ID NO: 159.

[0644] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes having IgM specific protease activity and one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 119. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzymes having IgM specific protease activity' and one or more disclosed recombinant enzymes comprising the sequence of any one SEQ ID NO: 119 - SEQ ID NO: 159. [0645] Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzy mes comprising the sequence of SEQ ID NO: 11 or SEQ ID NO: 12 and one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 1 19. Disclosed herein is a method of improving and/or enhancing transgene efficacy and/or expression the method comprising contacting one or more IgM molecules and/or IgG molecules with one or more disclosed recombinant enzy mes comprising the sequence of SEQ ID NO: 14 or SEQ ID NO: 15 and one or more disclosed recombinant enzymes comprising the sequence of SEQ ID NO: 119.

[0646] In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of SEQ ID NO: 119. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity⁷ to the sequence of SEQ ID NO: 1 19. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a recombinant enzyme comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of SEQ ID NO: 120 or SEQ ID NO: 121. In an aspect, a disclosed method can further comprise contacting one or more IgM molecules and IgG molecules with a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% identity to the sequence of any one of SEQ ID NO: 122 - SEQ ID NO: 159.

[0647] In an aspect, a disclosed method can further comprise administering one or more times one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0648] In an aspect, a disclosed method can further comprise measuring and/or determining a subject’s pre-treatment level of circulating IgG and/or IgM and/or a subject’s pre-treatment level of surface bound IgM. In an aspect, a disclosed method can further comprise measuring and/or determining one or more times a subject’s level of circulating IgG and/or IgM and/or a subject’s level of surface bound IgM (e.g., before, during, and after a disclosed administering step).

I. Kits

[0649] Disclosed herein is a kit comprising a disclosed enzy me, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or any combination thereof. Disclosed herein is a kit comprising one or more disclosed enzymes, one or more disclosed recombinant enzymes, one or more disclosed isolated nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0650] In an aspect, a disclosed kit can comprise one or more enzymes comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:46. In an aspect, a disclosed kit can comprise one or more enzy mes encoded by the sequence set forth in any one of SEQ ID NO:47 - SEQ ID NO:92. In an aspect, a disclosed kit can comprise one or more enzy mes encoded by the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. In an aspect, a disclosed kit can comprise one or more nucleic acid sequences comprising the sequence set forth in any one of SEQ ID NO:47 - SEQ ID NO: 92 and one or more nucleic acid sequences comprising the sequence set forth in SEQ ID NO: 160 or SEQ ID NO: 161. In In an aspect, a disclosed kit can comprise one or more recombinant enzy mes comprising the sequence set forth in any one of SEQ ID NO: 119 - SEQ ID NO: 159. In an aspect, a disclosed kit can comprise one or more recombinant enzymes having IgG specific protease activity’. In an aspect, a disclosed kit can comprise one or more recombinant enzymes comprising the sequence set forth in any one of SEQ ID NO:01 - SEQ ID NO:46 and one or more recombinant enzy mes comprising the sequence set forth in any one of SEQ ID NO: 119 - SEQ ID NO: 159.

[0651] In an aspect, a disclosed kit can comprise one or more plasmids (e.g., SEQ ID NO: 93).

[0652] In an aspect, a kit can comprise a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed RNA therapeutic, or a combination thereof, and one or more agents. ■'Agents" and ‘“Therapeutic Agents’" are known to the art and are described supra.

[0653] In an aspect, the one or more agents can treat, prevent, inhibit, and/or ameliorate one or more comorbidities in a subject. In an aspect, one or more active agents can treat, inhibit, prevent, and/or ameliorate cellular and/or metabolic complications related to a missing, deficient, and/or mutant protein, enzyme, or gene.

[0654] In an aspect, a disclosed kit can comprise at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose (such as, for example, treating a subject diagnosed with or suspected of having a disease or disorder). Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory’ device or downloaded from an internet website, or as recorded presentation. In an aspect, a kit for use in a disclosed method can comprise one or more containers holding a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed RNA therapeutic, or a combination thereof, and a label or package insert with instructions for use. In an aspect, suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers can be formed from a variety of materials such as glass or plastic. The container can hold a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, or a combination thereof, and can have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert can indicate that a disclosed enzyme, a disclosed recombinant enzyme, a disclosed isolated nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical formulation, a disclosed RNA therapeutic, or a combination thereof can be used for treating, preventing, inhibiting, and/or ameliorating a disease or disorder or complications and/or symptoms associated with a disease or disorder. A kit can comprise additional components necessary for administration such as, for example, other buffers, diluents, filters, needles, and syringes.

[0655] In an aspect, a disclosed kit can be used to cleave IgM and/or IgG, to cleave surface bound IgM on B cells, to reduce circulating levels of IgG and/or IgM, can inhibiting complement activation, can reduce and/or minimize vector mediated immunotoxicity and/or transgene immunogenicity, can improve and/or enhance transgene efficacy and/or expression. In an aspect, a disclosed kit can be used to effect these disclosed uses in a subject, such as, for example, a human subject.

J. Miscellaneous

[0656] Disclosed herein is a recombinant enzyme having human and non-human primate IgM specific protease activity and human and non-human primate IgG specific protease activity. In an aspect, a disclosed recombinant enzyme can comprise a sequence having at least 50% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. In an aspect, a disclosed recombinant enzyme can comprise a sequence having at least 50% identity to the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21. (IceMG2) In an aspect, a disclosed recombinant enzyme can cleave human IgM at the heavy chain at the interdomain region between constant domains Cp2 and Cp3. [0657] In an aspect, the IgM specific protease activity can occur between position 350 and position 365 of the IgM of SEQ ID NOTOS. In an aspect, a disclosed recombinant enzyme can cleave human or non-human primate IgG below the hinge region, thereby separating the Cp2 and Cp3 domains from the Cpl domain. In an aspect, the IgG specific protease activity can occur between positions 116 through 122 of the IgG of SEQ ID NO: 106, positions 112 through 118 of the IgG of SEQ ID NO: 107, positions 163 through 169 of the IgG of SEQ ID NO: 108, positions 113 through 119 of IgG of SEQ ID NO: 109, or any combination thereof. In an aspect, the IgM specific protease activity occurs at the sequence of SEQ ID NO: 110 of the IgM molecule, and wherein the IgG specific protease activity occurs at the sequence of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or any combination thereof of ahuman IgG molecule. [0658] Disclosed herein is an isolated nucleic acid molecule comprising a codon-optimized nucleic acid sequence encoding a disclosed recombinant enzyme. In an aspect, a disclosed isolated nucleic acid molecule can comprise the sequence set forth in SEQ ID NO:63 or SEQ ID NO:64. In an aspect, a disclosed isolated nucleic acid molecule can comprise the sequence set forth in SEQ ID NO:66 or SEQ ID NO:67. Disclosed herein is a recombinant enzyme having human and non-human primate IgM specific protease activity. In an aspect, a disclosed recombinant enzyme can comprise a sequence having at least 50% identity to the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. In an aspect, a disclosed recombinant enzy me can comprise a sequence having at least 50% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15. In an aspect, a disclosed recombinant enzyme can cleave the heavy chain at the interdomain region between constant domains Cp2 and Cp3 of human or non-human primate IgM. In an aspect, the IgM specific protease activity can occur between position 350 and position 365 of the IgM of SEQ ID NO: 105. In an aspect, the IgM specific protease activity can occur at the sequence of SEQ ID NO: 110 of an IgM molecule.

[0659] Disclosed herein is an isolated nucleic acid molecule comprising a codon-optimized nucleic acid sequence encoding a disclosed recombinant enzyme. In an aspect, a disclosed isolated nucleic acid molecule can comprise the sequence set forth in SEQ ID NO:57 or SEQ ID NO:58. In an aspect, a disclosed isolated nucleic acid molecule can comprise the sequence set forth in SEQ ID NO:60 or SEQ ID NO:61.

[0660] Disclosed herein is a pharmaceutical formulation comprising a disclosed recombinant enzyme.

[0661] Disclosed herein is a method of reducing the level of circulating IgM or the levels of circulating IgM and IgG in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a disclosed recombinant enzyme, wherein following the administering step, the level of circulating IgM or the levels of circulating IgM and IgG is reduced when compared to a pre-administering step level. In an aspect, the reduction of the level of circulating IgM or the levels of circulating IgM and IgG can be transient. In an aspect, the transient reduction of the level of circulating IgM or the levels of circulating IgM and IgG can persist about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, or more than 2 weeks. In an aspect, transient reduction of circulating level of IgM or the levels of circulating IgM and IgG can comprise at least an 80% reduction when compared to the pre-administering step level. In an aspect, a disclosed subject has received, is receiving, or will receive a recombinant product. In an aspect, a disclosed subject has received, is receiving, or will receive enzyme replacement therapy or protein replacement therapy. In an aspect, a disclosed subject has received, is receiving, or will receive one or more transplanted organs. In an aspect, following the administering step, the severity of a rejection event of the one or more transplanted organs can be reduced and/or minimized. In an aspect, a disclosed subject can have an autoimmune disease or disorder. In an aspect, a disclosed subject has received, is receiving, or will receive gene therapy.

[0662] Disclosed herein is a method of inhibiting the activation of the classical complement pathway in a subject, the method comprising: administering to the subject in need thereof a therapeutically effective amount of a disclosed recombinant enzyme, wherein following the administering step, the formation of the Cl and/or C3 complex is diminished and/or prevented.

[0663] Disclosed herein is a method of reducing and/or minimizing an immune response, the method comprising: administering to the subject in need thereof a therapeutically effective amount of a disclosed recombinant enzyme, wherein following the administering step, the level of circulating IgM or the level of circulating IgM and IgG targeting a recombinant product is reduced when compared to a pre-administering step level.

[0664] Disclosed herein is a method of reducing and/or minimizing an immune response, the method comprising: administering to the subject in need thereof (i) a therapeutically effective amount of a disclosed recombinant enzyme having IgM specific protease activity or IgM and IgG specific protease activity’ and (ii) a therapeutically effective amount of a recombinant enzyme having the sequence set forth in any of SEQ ID NO: 119 - SEQ ID NO: 159, wherein following the administering step, the levels of circulating IgM and IgG targeting a recombinant product is reduced when compared to a pre-administering step level.

[0665] In an aspect, a disclosed method can further comprise re-dosing the subject with the recombinant product. In an aspect, a disclosed recombinant product can comprise a vector or portion thereof. In an aspect, a disclosed recombinant product can comprise an encoded transgene product. In an aspect, a disclosed recombinant product can comprise a therapeutic protein or a therapeutic enzyme. In an aspect, a disclosed method can further comprise reducing and/or minimizing immunogenicity to the recombinant product. In an aspect, a disclosed method can further comprise improving and/or enhancing transgene efficacy and/or expression in a subject. In an aspect, transgene efficacy and/or expression in the subject can be improved and/or enhanced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% when compared to a pre-administering step level. [0666] Table 3 - Table of Sequences

VIII. EXAMPLES

[0667] Bacteria have evolved specific enz mes such as the IgG cleaving enzyme. IdeS from S. Pyogenes, to evade opsonization and immune recognition (Von Pawel-Rammingen U. et al. (2002) EMBO J. 21: 1607; Hulting G, et al. (2009) FEMS Microbiol Lett. 298:44-50; Lannergard

J, et al. (2006) FEMS Microbiol Lett. 262:230-235). These secreted enzymes belong to the papain-like cysteine protease superfamily and selectively cleave IgG at the Fc hinge region (Wenig K, et al. (2004) Proc Natl Acad Sci USA. 101 : 17371-17376).. However, no human IgM degrading enzymes have been reported to date. The work described below characterizes such enzymes.

Materials and Methods

1. IceM Structural Modeling and IceM-IgM Complex Analyses

[0668] Structural models for IceM and IceMG were generated using AlphaFold2-ptm (Jumper J, et al. (2021) Nature. 596(7873):583-589). Molecular graphics and analyses were performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases (Pettersen EF, et al. (2021) Protein Sci. 30(l):70-82). Structural models of IceM-IgM and IceMG-IgM/IgG complexes were generated using AlphaFol d2-multimer v2 using the existing structure of IdeS (PBD 8A47) as a template (Wenig

K, et al. (2004) Proc Natl Acad Sci USA. 101: 17371-17376). Contact residues analyses were performed using the select contacts command in ChimeraX with atomic distance < 5 Ang. For image rendering of complexes, superposition of AlphaFold2 prediction to PDB 2RCJ was performed using the matchmaker command in ChimeraX (Perkins SJ, et al. (1991) J Mol Biol. 221(4):1345-1366). 2. Plasmid Constructs and Recombinant Protein Expression

[0669] For expression of recombinant His-tagged Idessuis, IceM, or IceMG, coding sequences were synthesized by Twist Bioscience and cloned into the pET-21a expression vector. E. coli strain BL21 star was transformed with recombinant IceM-pET-21a or IceMG-pET-21a. A single colony was inoculated into Terrific Broth (TB) medium containing ampicillin and incubated in 37 °C at 200 rpm. When the OD600 reached ~4, the bacterial culture was induced with 1 mM isopropyl-P-d-thiogalactoside at 37 °C for 4 hr. Cells were harvested by centrifugation and lysed with lysis buffer (50 mM Tris-HCl, 150 mMNaCl, 1% TritonX-100, cocktail, pH 8.0). The target protein was obtained by 2-step purification. Proteins were purified from cell lysate supernatant using Ni-NTA resin followed by Superdex 200 column purification. Recombinant protein was stored in 50 mM Tris-HCl, 150 mM NaCl, and 10% glycerol, pH 8.0 and sterilized by 0.22 pm filter before being stored in aliquots. Endotoxin was removed from recombinant protein using High-Capacity Endotoxin Removal Spin Columns following the manufacturer’s instructions (Thermo Fisher Scientific, 88274). The concentration was determined by Bradford protein assay with BSA as standard. The protein purity and molecular weight of IceM (41.6 kDa) and IceMG (70.4k Da) were determined by standard SDS-PAGE followed by Coomassie staining with 2 pg BSA used as a standard.

3. Tissue Culture

[0670] Human peripheral blood mononuclear cells (ATCC, PCS-800-011) or Daudi cells (ATCC, CCL-213) were maintained at 37 °C and 5% CO2 in RPMI 1640 medium (Gibco, 11875119) supplemented with 10% fetal bovine serum (MilliporeSigma. F0926) according to vendor instructions.

4. SDS-PAGE and Western Blot Analysis

[0671] Samples were incubated at 95 °C for 5 min in LDS loading buffer supplemented with 2.5% P-Mercaptoethanol, separated by SDS-PAGE, and either stained using coomassie blue (Themo Fisher Scientific, 24590) or transferred to PVDF membrane. Membranes were blocked with 5% milk powder in Tris buffered saline with 0.1 % TWEEN 20 (TBST) and incubated for 1 hr at room temperature with horseradish peroxi dase-conjugated primary antibodies for human IgM (1:5000; Proteintech, HRP-66484), porcine IgM (1:5000; Novus, NBP2-42699H), mouse IgM (1: 10,000; Invitrogen. 62-6820) IgG (1: 10.000; Invitrogen, 31423), IgA (1: 1000; Invitrogen. A18781) IgE (1 : 1000; Invitrogen, A18793), IgD (1 :5000; Proteintech, 16489-1-AP), or 6xHistidine (1 : 10,000; Proteintech, HRP-66005). Membranes were then washed 3x with TBST and incubated for 3 min with enhanced chemiluminescence substrate (Thermo Fisher Scientific, 34049). Coomassie- stained gels and immunoblots were visualized on a Bio-Rad ChemiDoc imaging system, and where applicable, quantitated using Bio-Rad Image Lab Version 6.1.0. 5. In Vitro Cleavage Assays

[0672] IgM cleavage was assessed by treating 10 jag of purified human IgM (MilliporeSigma 18260) with 10 pg mL’¹ IceM, IceMG, or PBS and incubated at 37 °C for 1 hr. Enzy matic reactions were halted by adding LDS loading buffer, separated by SDS-PAGE, and stained with Coomassie blue as described above. For serum samples, 25 pL of sera were treated with IceM at the concentration indicated for 1 hr at 37 °C. Sera samples were diluted 1 :10 or 1 :50 for IgM or IgG immunoblots, respectively. Western blots were completed as described above and probed using anti-IgM, anti-IgG, anti-IgA, anti-IgE, or anti-IgD antibodies. Human sera were purchased from BioIVT; pig, dog and mouse serum samples were obtained from in-house lab stocks. Nonhuman primate sera were gifts from Alice Tarantal (UCD, Davis, California, USA).

6. Microscopy and Immunofluorescence

[0673] Daudi cells incubated with PBS, recombinant IceM at the concentration indicated for 1 hr at 37 °C. Cells were washed 3x with ice cold PBS and then cytocentrifuged onto Lab-Tek II Chamber Slides (Thermo Fisher Scientific 154453) coated with Poly-L-lysine. Cells were washed with PBS supplemented with 1 mM CaCh and 1 mM MgCb and then fixed using 10% formalin for 30 min. Cells were blocked with 5% normal goal serum in PBS with for 30 min and then incubated with F(ab)-specific anti-human IgM antibody conjugated to FITC for 1 hr at room temperature. Following three washes with PBS and slides were mounted with Prolong Gold Antifade Reagent with DAPI (Invitrogen P36930). Immunofluorescence was visualized using 10X or 20X Olympus objectives on a Zeiss 880 Airyscan Fast Inverted Confocal microscope.

7. Flow Cytometry Analysis

[0674] For surface bound IgM cleavage analysis, 3e5 PBMCs or 5e4 Daudi cells were incubated with PBS, recombinant IceM, or recombinant IceMG at the concentration indicated for 1 hr at 37 °C. Cells were washed 3x with ice cold PBS and then incubated with F(ab)-specific anti-human IgM antibody conjugated to FITC (LSBio. LS-C536848), anti-CD19 conjugated to APC (Abeam, AB 18224), and Zombie Violet (BioLegend, 77477) to assess cell viability per manufacturer recommendations in PBS supplemented with 3% BSA on ice for 1 hr. Cells were then washed 3x with ice cold PBS and analyzed using a BD Biosciences Fortessa X-20 flow cytometer. Flow data were gated on live cells and then CD19⁺ (APC⁺) to analyze the IgM⁺ (FITC⁺) cohort. Experiments were performed in triplicate. Data were analyzed using FlowJo v. 10.8.1 (BD Biosciences) and graphs w ere generated using GraphPad Prism version 9.5.

8. Complement Assays

[0675] Complement activation assays were adapted from Khandelwal et al., 2018 (Blood. 132:2431-2440). To assess the effect of IceM on complement activation, 25 .L of complement preserved human sera (BioIVT, HUMANSRM-01017110) w ere incubated with PBS or 20 pg mL" ¹ of recombinant IceM, or recombinant IceMG for 1 hr at 37 °C. Samples were then treated with PBS, 5el0 vector genomes AAV9, 25 pg mL^-1:0.25 U mL'¹ Platelet Factor 4 (PF4):Heparin, or 10 mM EDTA + Platelet Factor 4 (PF4): Heparin for 1 hr at 37 °C. Complement activation was evaluated by assessing complement protein C3a levels by commercial ELISA kit (Thermo Fisher Scientific, BMS2089) and absorbance was read using a Victor X3 microplate reader (PerkinElmer) per manufacturer specifications.

9. In Vivo Mouse and Non-Human Primate Studies

[0676] All animal care and procedures were conducted in accordance with National Institutes of Health (NIH) guidelines and approved by the Duke University Institutional Animal Care and Use Committee (Duke IACUC# Al 86-22-10 and Al 13-21-05). For mouse studies, 12-wk-old female C57BL/6J mice were purchased from Jackson Laboratories and injected intraperitoneally with pooled 200 pL of pooled human sera (MilliporeSigma, H4522). After 24 hr, 200 pL of PBS or IceM was administered intravenously by tail vein injection at the dose indicated. Blood samples were taken by submental bleeds at 24 hr post-IceM injection, allowed to coagulate for 30 min, and serum was separated by centrifugation. Mouse sera was diluted 1 : 10 in LDS loading buffer with 2.5% [3-Mercaptoethanol, incubated at 95 °C for 5 min prior to SDS-PAGE, and western blots for anti-IgM were performed as described above. 5 mice (numbered 1-5) were tested per experimental condition. Human sera positive control was included with the PBS samples. For nonhuman primate studies, two male rhesus macaques (macaca mulatta) obtained from Alphagenesis, Inc (Y emassee, SC, USA), animal IDs N303 (6.7kg) and M741 (8.2 kg), were selected for the study. All animals received two doses of IceM at 0.5 mg kg^-1 (one month apart) to evaluate the effects of the IgM endopeptidase post-infusion. To assess enzyme activity, blood was collected on the following days: 0, 1, 4, 7, 14, 21, 28 post-IceM treatment. Animals were re-dosed on day 28 and blood was collected on the following days: 29, 32, 35, 42, and 49. NHP sera was diluted 1 : 10 in LDS loading buffer with 2.5% P-Mercaptoethanol, incubated at 95 °C for 5 min prior to SDS- PAGE, and western blots for anti-IgM, anti-IgG, and anti-6xHistidine were performed as described above. Western blots were quantitated using Bio-Rad Image Lab Version 6.1.0. Schematics w ere created with BioRender.com.

10. Statistical Analyses

[0677] Error bars denote mean ± standard deviation. All experiments were completed in at least biological triplicate. Statistical significance w as determined using unpaired, two-tailed Student’s /-test (FIG. 2B and FIG. 2F) or two-way ANOVA with Sidak’s correction for multiple comparisons (FIG. 2D). Analyses were performed with GraphPad Prism version 9.5 with P values less than 0.05 being significant. Example 1 Characterization of Candidate Human IgM Proteases

[0678] Candidate human IgM proteases were selected and evaluated. The core protease domain was identified and engineered to exclude elaborations such as cell wall binding and excretion motifs to enhance recombinant protein production and then expressed by an IPTG inducible promoter in E. coll (FIG. 1A). Of the candidates tested, two from Lachnoanaerobaciiltim saburreum (Protein 2; NCBI txid: 467210) and Lachnoanaerobaculum umeaense (Protein 6; NCBI txid: 617123) were identified as human IgM cleaving proteases (herein IceM and IceM2) that produced bands consistent with separation of Cp3 and Cp4 domains from the remainder of IgM heavy chain (FIG. IB - FIG. IE). IceM and IceM2 have 76% sequence identity excluding consideration of insertions (246/322 residues) and 75% identity when considering insertions (246/328 residues), within the portion synthesized for testing.

[0679] To further characterize enzymatic activity, recombinant IceM expression in E. coli and purification by Nickel column affinity resin was confirmed via SDS-PAGE (FIG. IE). Treatment of purified human IgM with IceM generated two cleavage products at 41 kDa and 32 kDa indicating hydrolysis of the IgM heavy chain at the interdomain region between constant domains Cp.2 and Cp3 consistent with predictive structural modeling (FIG. ID, FIG. IF). IceM efficiently and specifically cleaved IgM with an ECso of ~0.16 nM, but did not cleave isotypes IgG, IgA, IgE or IgD (FIG. 1G, FIG. 1H) Further, IceM cleaves human and non-human primate IgM in vitro, but not IgM from other species such as dog, pig, or mouse (FIG. II). This high level of specificity is due to specific residues Asp 355, Thr 356, Ala 357, He 358, Arg 359, and Vai 360 (amino acid numbering based on IgM Sequence in Accession No. QBK47415.1) that surround the cleavage site and are unique to human and nonhuman primate IgM (FIG. 1 J).

[0680] Recently the structure of IdeS. another highly specific IgG cleaving papain-like protease superfamily member, was determined in complex with human IgG and demonstrated that human IgG binds to a major pocket adjacent to the enzyme active site (Sudol A S L, et al. (2022) Nature Commun. 13: 1-11) . Next, the high specificity of IceM for IgM was examined. Utilizing AlphaFold2, the structure of this human IgM cleaving enzymes in complex with two human IgM heavy chains was predicted, and then the best ranked structure was used for the interpretation. In the determined structures, the the human IgM heavy chains were bind within the same pocket as determined for the IdeS/IgG Complex (FIG. 2A - FIG. 2C). As a result, the specificity of these enzymes for human IgM w as determined to be due to differences in amino acid composition of the major binding pocket adjacent to the cleavage site for specific Igs. Further, absolute placement of this Ig domain likely contributes a major factor in the specific cleavage product observed within the Ig hinge. Similar amino acid positions were responsible for human IgM binding in the outer active site clefts of both IgM proteases (FIG. 2B, FIG. 2C). Of residues that directly interact within 5 Angstroms of the IgM, 76% identity was observed among residues in common between both IceM and IceM2 (51/67 residues); among all contact residues 70% identity between both was observed, where inserted residues were counted as mismatched (65/93 residues) (Table 1). In Table 1. the list of amino acid residues in the outer active site clefts of IceM and IceM2 that directly interact within 5 Angstroms of the corresponding human IgM residue (as represented in SEQ ID NO: 105 // QBK47415.1). Homologous positions from IceM and IceM2 are indicated in bold. These numbers were lower than for the proteins overall, likely because enzyme core domains generally are more conserved than enzyme surface.

[0681] Table 1 - Contact Residues of IceM and IceM2 to Human IgM.

Example 2 Construction of Recombinant Fusion Protein

[0682] Next, various linkers varying in length and flexibility were designed to engineer a recombinant fusion protein comprising IceM and IdeZ proteolytic domains (FIG. 3A, top). Plasmids for three different fusion protein linkers (rigid linker, flexible linker 1 , and flexible linker 2) were each constructed and expressed using E. coli. After incubation, bacteria were collected, lysed, and pre- and post-induction samples were analyzed by western blot for anti-His. Significantly greater protein expression for the rigid linker was observed when compared to the flexible linkers indicated by the bands at approximately 70 kDa (FIG. 3A, bottom). Enzy mesubstrate docking analyses indicated the rigid fusion protein, hereinafter termed IceMG, maintained the ability to tightly bind human IgM and IgG within their respective catalytic pocket (FIG. 3B, FIG. 3C) Recombinant IceMG expression in E.Coli and purification by Nickel column affinity resin was confirmed via SDS-PAGE (FIG. 4A). Treatment of purified human IgM with IceMG generated two cleavage products at 41 kDa and 32 kDa consistent with hydrolysis of the IgM heavy chain at the interdomain region between constant domains Cp2 and Cp.3 and with predictive structural modeling of previous IceM cleavage analysis (FIG. 4B). Treatment of purified human IgG with IceMG generated cleavage products at 25 kDa slightly above the light chain representing hydrolysis at a single site below the hinge region, yielding F(ab')2 and Fc fragments, consistent with prior reports of IdeZ (Hulting G, et al. (2009) FEMS Microbiol Lett. 298:44-50). . Treatment of IceMG efficiently and specifically cleaved IgM and IgG with an EC so of -0.07 and 4.7 nM, respectively, but did not cleave isotypes IgA, IgE, IgD (FIG. 4E -FIG. 4G). Further, IceMG cleaved human and non-human primate IgM and IgG in vitro, but not IgM or IgG from other species such as dog, pig, or mouse (FIG. 4H). For IceMG, the specificity for IgG is listed in FIG. 4J and detailed in Table 2 with amino acid numbering based on sequence accession number noted for each subtype. In Table 2, amino acid sequences for IgG subtypes with the cleavage site indicated by backslash. Specific IgG residues immediately within the cleavage are listed. Amino acid numbering is relative to the IgG sequence accession number noted.

[0683] Table 2 - Critical Amino Acid Residues Within the Cleavage Site for IgGl, IgG2, IgG3, and IgG4.

Example 3 Removal of Surface Bound IgM From B Cells

[0684] The ability for IceM and IceMG to remove surface-bound IgM (BCR) from B cells was examined. IceM completely removed IgM from the surface of the B lymphoblast cell line Daudi within 1 hr (FIG. 5A FIG. 5D). Further, IceM robustly cleaved IgM BCR from the surface of CD19⁺ sub-population in human peripheral blood mononuclear cells (PBMCs) at an EC 50 ~70 nM (FIG. 5E - FIG. 5G). Importantly, surface IgM baseline levels were restored ~12 hr posttreatment - indicating that BCR inactivation is transient and reversible (FIG 5E, FIG. 5F) with no adverse impact on cell viability after 24 hr (97.9% and 97.8% for PBS-treated PBMCs and IceMtreated PBMCs, respectively). Given that IgM is a potent activator of the classical complement pathway triggered by binding Clq, the ability of IceM and IceMG to block complement activation was examined. Using Adeno-associated virus 9 or an in vitro platelet factor-based assay (Khandelwal S, et al. (2018) Blood. 132:2431-2440) mimicking heparin induced thrombocytopenia (FIG. 6A), the pre-treatment of human sera with IceM, and to greater extent, IceMG was determined to block the IgM-triggered complement cascade at levels comparable to EDTA (FIG. 6B, FIG. 6C). These unique properties of IceM and IceMG corroborate the notion that selective IgM cleavage can potentially be leveraged to inactivate B cell sensing and signaling leading to antigen-induced Ig class switching as well as blocking complement activation.

Example 4 Clearance of IgG in Human Sera

[0685] To evaluate Ig clearance in vivo, mice were passively immunized w ith pooled human sera and w ere then administered IceM or IceMG intravenously at doses of 0.25 mg kg’¹, 1.0 mg kg’¹, or 2.5 mg kg’¹ (FIG. 7A). Rapid (within 24 hr) and near-complete clearance of circulating human IgM and IgG at all doses was observed when compared to control animals for IceM (FIG. 7B - FIG. 7E) and IceMG (FIG. 7F - FIG. 71). Based on these results, whether pretreatment wdth IceM or IceMG could rescue AAV transduction in mice passively immunized with human sera and IVIg containing neutralizing antibodies in vivo was evaluated (FIG. 7 J). In animals treated with only sera/IVIg, approximately a log decrease in AAV8-mediated(FIG. 7K, FIG. 7L) and AAV9- (FIG. 7M, FIG. 7N) mediated luciferase expression in heart and liver tissue was observed. In animals treated wdth IceM, a partial rescue in transduction from neutralizing antibodies was observed. In animals treated with IceMG, a complete rescue of luciferase expression in heart and liver tissue was observed (FIG. 7K - FIG. 7N). Interestingly, serotypespecific differences in the level of rescue for animals treated with IceM were observed. These differences could likely be due to timing at which the serum donor was exposed to AAV or seroty pe-specific differences in prevalence or neutralizing ability⁷ of IgM. The ability⁷ of IceM and IceMG to effectively degrade anti-AAV neutralizing antibodies in vivo may present a new paradigm for expanding the patient cohort, reducing vector mediated immunotoxicity and potential redosing of AAV based gene therapies.

Example 5 Evaluation of Protease Activity in Non-Human Primates

[0686] These enzy mes were next evaluated in nonhuman primates in vivo. First, two rhesus macaques (animal ID: N303 & M741) were dosed with recombinant IceM at a dose of 0.5 mg kg" ¹ and the kinetics of IgM clearance in serum were monitored at different time intervals (FIG. 8A). Pharmacokinetics of IceM in monkey sera revealed rapid clearance of the recombinant enzyme, with detectable levels at day 1 post-administration, but not subsequent time intervals (FIG. 8B, FIG. 8C). N303 and M741 exhibited 95.4 % and 91.4 % IgM clearance, respectively, at 24 hr post-administration with restoration of baseline levels within ~2 weeks post-treatment (FIG. 8D - FIG. 8F). In addition, repeat dosing of IceM at day 28 yielded similar results with no loss in IgM cleavage activity or any detectable change in pharmacokinetics (FIG. 8D - FIG. 8F).

Example 6 Characterization of Kinetics in Non-Human Primates

[0687] Next, ine macaques were dosed in two separate studies (animal ID: 49873, 49813, 49682, 36725, 37662, 35788, 38131, 38718, 38721) at 0.25 mg kg ¹, 0.5 mg kg ¹, 1.0 mg kg , or 2.5 mg kg"¹ IceMG, and then monitored for kinetics of IgM and IgG clearance at the time intervals indicated (FIG 9A and FIG. 10A). Pharmacokinetics of IceMG in monkey sera revealed rapid, clearance of the recombinant enzyme, with detectable levels up to 24 hr post-administration, but not subsequent time intervals (FIG. 9B - FIG. 9D, top panel). Majority of recombinant IceM was cleared between 6 hr and 24 hr post-administration. Animals exhibited near complete clearance of circulating IgM in both studies with IgM levels returning close to or at baseline between 7-14 days post-treatment (FIG. 9B - FIG. 9G, middle panel; FIG. 10B FIG. 10G, top panel). IgG levels for most animals exhibited significant, yet not complete clearance FIG. 10A - FIG. 10F). Notably, complement activation elicited by AAV9 was blocked in vitro in sera from animals 49873, 49813, 49682 following pre-treatment with IceMG (FIG. 9E). These results corroborated the high degree of selectivity and robust enzymatic cleavage of primate IgM and IgG by these enzy mes in vivo.

Summary of Examples

[0688] The Examples describe highly specific endoproteases that can rapidly cleave human and NHP IgM or both IgM and IgG. The data demonstrate the ability to enzymatically remove IgM BCR in vitro and degrade circulating IgM in vivo in both mice and nonhuman primates. Notably, pretreatment with IceM and IceMG prevented complement activation in human and nonhuman primate sera which may have therapeutic implications. One example would be kidney transplant recipients who are sensitized to human leukocyte antigens (HLAs) expressed in the transplanted organ have significantly increased rates of acute antibody mediated rejection (AMR) (Lefaucheur C, et al. (2010) J Am Soc Nephrol. 21 : 1398-1407; Bentall A, et al. (2013) Am J Transplant. 13: 76- 85). These donor-specific antibodies cause acute AMR largely through the activation of the complement cascade (Stegall M D, et al. (2012) Nat Rev Nephrol. 8:670-678). IgM is the predominant driver of complement activation and increased donor-specific IgM levels has been associated with organ rejection (Schmitz R, et al. (2021) Nat Commun. 12(1): 5456). Therefore, targeting donor-specific IgM antibodies using IceM in concert with current strategies may represent a viable option to further decrease AMR in sensitized transplant recipients.

[0689] Another example where targeting IgM would be appealing is AAV-mediated gene therapy. While it has been well known that preexisting and neutralizing antibodies prevent the broader application of therapeutic gene transfer, recent clinical trials have show n that antibody response and complement activation can lead to serious adverse events including death, particularly in high dose cohorts (Ertl HCJ, et al. (2022) Front Immunol. 13:985382; Shen W, et al. (2022) Front Immunol. 13: 1001263) . Further, it is well-documented that AAV administration triggers a swift and robust rise in IgM, that after class switching, can lead to immune memory' (Gross DA, et al. (2022) Front Immunol. 13:857276). Therefore, targeting neutralizing antibodies and the complement cascade with IceM and IceMG may decrease toxicity associated with complement activation and may enable AAV redosing by addressing memory immune response.

[0690] The degradation of IgM and IgG could also have profound implications in autoimmune disease as well. It is estimated that over 2.5% of the world’s population has an autoantibody driven autoimmune disease (Edna Au et al., Frontiers in Med. 2022). More specifically, elevated levels of IgM and IgG autoantibodies are associated with multiple autoimmune diseases including systemic lupus ery thematosus, rheumatoid arthritis and multiple sclerosis just to name a few'. Currently, there are no available therapeutics that directly address pathogenic IgM antibodies.

[0691] To this end, IceM and IceMG provide a powerful therapeutic solution with 3 key defining characteristics. First, IceM and IceMG cleared IgM and IgG from circulation in vivo. Second, IceM and IceMG were able to inactivate the antibody driven classical complement pathway by clearing IgM and IgG antibody complexes deposited on tissues such as the kidney. Lastly, IceM and IceMG effectively cleave the IgM BCR providing a potential solution to prevent the generation of class switched IgG autoantibodies.

[0692] Taken together, IceM and IceMG are promising candidates for modulating humoral immunity' in primates and therapeutic removal of IgM or both IgM and IgG in pathophysiological settings that may present in autoimmune disorders, organ transplantation, and AAV gene therapy.

Claims

IX. CLAIMS What is claimed is:

1. A recombinant enzyme having human and non-human primate IgM specific protease activity and human and non-human primate IgG specific protease activity⁷.

2. The recombinant enzyme of Claim 1 comprising a sequence having at least 50% identity to the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18.

3. The recombinant enzyme of Claim 1 comprising a sequence having at least 50% identity to the sequence set forth in SEQ ID NO:20 or SEQ ID NO:21.

4. The recombinant enzyme of any one of Claims 1 - 3, wherein the recombinant enzy me cleaves human IgM at the heavy chain at the interdomain region between constant domains Cp2 and Cp3.

5. The recombinant enzyme of any⁷ one of Claims 1 - 4, wherein the IgM specific protease activity⁷ occurs between position 350 and position 365 of the IgM of SEQ ID NO:105.

6. The recombinant enzyme of any one of Claims 1 - 5, wherein the recombinant enzyme cleaves human or non-human primate IgG below the hinge region, thereby separating the Cp2 and Cp3 domains from the Cpl domain.

7. The recombinant enzy me of any one of Claims 1 - 6, wherein the IgG specific protease activity⁷ occurs between positions 116 through 122 of the IgG of SEQ ID NO: 106, positions 112 through 118 of the IgG of SEQ ID NO: 107, positions 163 through 169 of the IgG of SEQ ID NO: 108, positions 113 through 119 of IgG of SEQ ID NO: 109, or any combination thereof.

8. The recombinant enzy me of any one of Claims 1 - 6, wherein the IgM specific protease activity⁷ occurs at the sequence of SEQ ID NO: 110 of the IgM molecule, and wherein the IgG specific protease activity occurs at the sequence of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 1 13, SEQ ID NO: 1 14, or any combination thereof of a human IgG molecule.

9. An isolated nucleic acid molecule comprising a codon-optimized nucleic acid sequence encoding the recombinant enzy me of any one of Claims 1 -8.

10. The isolated nucleic acid molecule of Claim 9 comprising the sequence set forth in SEQ ID

NO:63 or SEQ ID NO:64.

11. The isolated nucleic acid molecule of Claim 9 comprising the sequence set forth in SEQ ID

NO: 66 or SEQ ID NO: 67.

12. A recombinant enzyme having human and non-human primate IgM specific protease activity.

13. The recombinant enzyme of Claim 12 comprising a sequence having at least 50% identity to the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12. recombinant enzyme of Claim 12 comprising a sequence having at least 50% identity to the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15. recombinant enzyme of any one of Claims 12 - 14, wherein the recombinant enzy me cleaves the heavy chain at the interdomain region between constant domains Cp2 and CLI3 of human or non-human primate IgM. recombinant enzyme of any one of Claims 12 - 15, wherein the IgM specific protease activity⁷ occurs between position 350 and position 365 of the IgM of SEQ ID NO: 105. recombinant enzyme of any one of Claims 12 - 15, wherein the IgM specific protease activity occurs at the sequence of SEQ ID NO: 110 of an IgM molecule. isolated nucleic acid molecule comprising a codon-optimized nucleic acid sequence encoding the recombinant enzy me of any one of Claims 12 - 17. isolated nucleic acid molecule of Claim 18 comprising the sequence set forth in SEQ ID

NO:57 or SEQ ID NO:58. isolated nucleic acid molecule of Claim 18 comprising the sequence set forth in SEQ ID

NO:60 or SEQ ID NO:61. harmaceutical formulation, comprising: the recombinant enzyme of any one of Claims 1 -

8 or Claims 12 - 17. ethod of reducing the level of circulating IgM or the levels of circulating IgM and IgG in a subject, the method comprising: administering to the subject in need thereof a therapeutically effective amount of the recombinant enzyme of any one of Claims 1 - 8 or Claims 12 - 17, wherein following the administering step, the level of circulating IgM or the levels of circulating IgM and IgG is reduced when compared to a pre-administering step level. method of Claim 22, wherein the reduction of the level of circulating IgM or the levels of circulating IgM and IgG is transient. method of Claim 23. wherein the transient reduction of the level of circulating IgM or the levels of circulating IgM and IgG persists about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 2 weeks, or more than 2 weeks. method of Claim 24, wherein the transient reduction of circulating level of IgM or the levels of circulating IgM and IgG comprises at least an 80% reduction when compared to the pre-administering step level. method of any one of Claims 22 - 25, wherein the subject has received, is receiving, or will receive a recombinant product. method of any one of Claims 22 - 25, wherein the subject has received, is receiving, or will receive enzyme replacement therapy or protein replacement therapy. method of any one of Claims 22 - 25, wherein the subject has received, is receiving, or will receive one or more transplanted organs. method of Claim 28. wherein, following the administering step, the severity of a rejection event of the one or more transplanted organs is reduced and/or minimized. method of any one of Claims 22 - 25, wherein the subject has an autoimmune disease or disorder. method of any one of Claims 22 - 25, wherein the subject has received, is receiving, or will receive gene therapy. ethod of inhibiting the activation of the classical complement pathway in a subject, the method comprising: administering to the subject in need thereof a therapeutically effective amount of the recombinant enzymes of any one of Claims 1 - 8 or Claims 12 - 17, wherein following the administering step, the formation of the Cl and/or C3 complex is diminished and/or prevented. ethod of reducing and/or minimizing an immune response, the method comprising: administering to the subject in need thereof a therapeutically effective amount of the recombinant enzyme of any one of Claims 1 - 8 or Claims 12 - 17, wherein following the administering step, the level of circulating IgM or the level of circulating IgM and IgG targeting a recombinant product is reduced when compared to a pre-administering step level. ethod of reducing and/or minimizing an immune response, the method comprising: administering to the subject in need thereof (i) a therapeutically effective amount of the recombinant enzyme of any one of Claims 1 - 8 or Claims 12 - 17 and (ii) a therapeutically effective amount of a recombinant enzy me having the sequence set forth in any of SEQ ID NO: 119 - SEQ ID NO: 159, wherein following the administering step, the levels of circulating IgM and IgG targeting a recombinant product is reduced when compared to a pre-administering step level. method of Claim 33 or Claim 34, further comprising re-dosing the subject with the recombinant product. method of Claim 33 or Claim 34, wherein the recombinant product comprises a vector or portion thereof. method of Claim 33 or Claim 34, wherein the recombinant product comprises an encoded transgene product. method of Claim 33 or Claim 34, wherein the recombinant product comprises a therapeutic protein or a therapeutic enzyme. method of any one of Claims 33 - 38, further comprising reducing and/or minimizing immunogenicity to the recombinant product. method of Claim 37, further comprising improving and/or enhancing transgene efficacy and/or expression in a subject. method of Claim 40, wherein transgene efficacy and/or expression in the subject is improved and/or enhanced by about 10%, 20%, 30%, 40%, 50%, 60%. 70%, 80%, 90%, 100%, or more than 100% when compared to a pre-administering step level.