RU2825840C2 - Gene therapy monitoring - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a group of inventions comprising a construct for use in measuring expression of a therapeutic payload and a method for monitoring gene therapy. Construct comprises 5' to 3' polynucleotide sequence coding (a) 5'-homologous arm, (b) coding sequence 2A, coding peptide 2A, where coding sequence 2A contains a nucleotide sequence coding the amino acid sequence SEQ ID NO: 1, (c) a therapeutic payload and (d) 3'-homologous arm, wherein said construct is integrated into an endogenous albumin target site so that the albumin locus is transcribed to form albumin-2A and a therapeutic payload in the form of separate proteins.

EFFECT: invention extends the range of agents for modifying the expression of the therapeutic payload.

20 cl, 17 dwg, 11 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/833,875, filed April 15, 2019, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LIST

[0002] This application contains a Sequence Listing, which has been submitted electronically as a .txt file named "2012538-0082_SL.txt". The .txt file was created on April 10, 2020 and is 26,104 bytes in size. The entire contents of the Sequence Listing are incorporated herein by reference.

LEVEL OF TECHNOLOGY

[0003] There are a number of human diseases that can be linked to changes in DNA that are either inherited or acquired early in embryonic development. Of particular interest to developers of genetic therapy (also called "gene therapy") are diseases caused by a mutation in a single gene, known as monogenic diseases. There are thought to be over 6,000 monogenic diseases. Generally, each specific genetic disease caused by inherited mutations is relatively rare, but collectively the number of diseases linked to genetics is large. Notable genetic diseases include cystic fibrosis, Duchenne muscular dystrophy, Huntington's disease, and sickle cell anemia. Other classes of genetic diseases include metabolic disorders such as organic acidemias and lysosomal storage diseases, where dysfunctional genes lead to defects in metabolic processes and accumulation of toxic byproducts that can lead to significant morbidity and mortality in both the short and long term.

[0004] While gene therapy has received much attention in terms of developing therapeutic candidates, much less attention has been paid to methods for monitoring and evaluating the effectiveness and trajectory of gene therapy. New methods and compositions are needed to ensure the effective use of gene therapy, including in combination with one or more additional treatments.

The essence of the invention

[0005] The present disclosure provides, among other things, technologies for improving gene therapy. Among other things, the present disclosure provides technologies for monitoring and/or assessing one or more characteristics of a gene therapy treatment, such as, for example, the extent, level, and/or persistence of payload expression. In some embodiments, the disclosed technologies are particularly useful in integrative gene therapy.

[0006] Among other things, the present disclosure demonstrates that certain integrative gene therapy technologies can generate new biomarkers whose expression and/or activity can be highly correlated with the expression and/or activity of a payload of interest (e.g., a product encoded by and/or expressed from a transgene) delivered by the gene therapy. Moreover, according to the present disclosure, such generation can provide strategies for monitoring and/or otherwise assessing the gene therapy and/or its success, stability, maintenance, etc.

[0007] Among other things, in some embodiments of the invention, it has been discovered that the currently disclosed biomarkers can be assessed directly from a biological sample collected non-invasively from a subject receiving gene therapy, and that assessment of such biomarkers can provide information about the state of the payload that would otherwise require more invasive procedures to determine. As one example, the present disclosure demonstrates that a tissue biopsy is not required to determine the delivery or expression of the payload in a tissue. Rather, analysis of biomarkers from the circulation (e.g., via a blood draw) can be assessed to indirectly detect one or more aspects of the expression and/or activity of the payload in the tissue. This can simplify and facilitate analysis of the payload delivered to intracellular locations.

[0008] In some embodiments, the present disclosure provides methods for monitoring gene therapy, the methods comprising the step of detecting in a biological sample from a subject that has received an integration gene therapy a level or activity of a biomarker created as a result of the integration of the integration gene therapy as a surrogate for one or more characteristics of the status of the gene therapy treatment, wherein the one or more characteristics of the status of the gene therapy treatment is selected from the group consisting of a payload level, a payload activity, a level of integration of the gene therapy treatment into a population of cells, and combinations thereof.

[0009] In some embodiments, the present disclosure provides methods for monitoring the delivery, level, and/or activity of a payload in a subject that has received a gene integration composition that delivers the payload, the methods comprising the step of detecting in a biological sample of the subject a level or activity of a biomarker created as a result of the integration of the gene integration composition, as a surrogate for the delivery, level, and/or activity of the payload.

[00010] In some embodiments, the present disclosure provides methods for determining one or more characteristics of gene therapy treatment status in a subject that has received treatment with an integrative gene therapy agent, the methods comprising the steps of: a) obtaining a biological sample from the subject, b) determining a level of a biomarker, wherein the biomarker is generated as a result of integrative gene therapy into the subject's genome, and c) based on the determined level of the biomarker, establishing one or more characteristics of gene therapy treatment status in the subject, wherein the determined level of the biomarker corresponds to the one or more characteristics of gene therapy treatment status.

[00011] In some embodiments, the present disclosure provides methods for delivering a gene therapy treatment to a subject in need thereof, including the steps of a) administering an integrative gene therapy treatment to the subject and b) determining in a biological sample of the subject the level of a biomarker that results from the integration of the gene therapy treatment into the subject's genome.

[00012] In some embodiments, treatment with an integrative gene therapy agent or a gene integration composition provides for the integration of a nucleic acid element comprising a sequence that encodes a payload into a target site in the genome of a subject. Those skilled in the art will appreciate that any of a variety of target sites may be suitable for use with the methods and compositions described herein. For example, in some embodiments, the target site encodes a polypeptide (e.g., albumin). In some embodiments, the integration of the nucleic acid element occurs at the 5' or 3' end of the gene that encodes the polypeptide. In some embodiments, the target site encodes albumin.

[00013] According to various embodiments of the invention as described herein, any payload suitable for a particular application can be used. In some embodiments, the payload is or consists of a peptide/polypeptide/protein, a nucleic acid (e.g., shRNA, microRNA), and any combination thereof. For example, in some embodiments, the payload is or includes a peptide expressed intracellularly. In some embodiments, the payload is or includes a peptide secreted intracellularly. In some embodiments, the payload is a peptide that has intracellular or extracellular activity that promotes a biological process for treating a medical condition. In some embodiments, the payload is a peptide that is normally expressed in liver cells. In some embodiments, the payload is a peptide that is ectopically expressed in liver cells. In some embodiments, the payload is methylmalonyl-CoA mutase, alpha-1-antitrypsin, or human factor IX.

[00014] As described herein, many embodiments include the use of one or more biological samples (e.g., a fluid or tissue sample taken from a subject). In accordance with the present disclosure, any of a plurality of biological samples is considered compatible with various embodiments. For example, in some embodiments, the biological sample is or includes hair, skin, stool, blood, plasma, serum, cerebrospinal fluid, urine, saliva, tears, vitreous humor, or mucus.

[00015] As described herein, detection (e.g., detection of a signal, such as a biomarker or a detectable fragment) in connection with the methods and compositions described herein can be achieved by any method suitable for use. For example, in some embodiments, the detection step is or includes an immunoassay or a nucleic acid amplification assay.

[00016] According to the present disclosure, the use of a plurality of biological samples is considered consistent with various embodiments. In some embodiments, the biomarker is or includes a detectable fragment that is fused to the polypeptide encoded by the target site upon translation of the polypeptide encoded by the target site. In some embodiments, the biomarker is or includes a detectable fragment that is fused to the polypeptide encoded by the target payload upon translation of the polypeptide encoded by the target site. In some embodiments, the biomarker is or includes a 2A peptide. In some embodiments, the 2A peptide is selected from the group consisting of P2A, T2A, E2A, and F2A. In some embodiments, the biomarker can be or consist of a furin cleavage motif. In some embodiments, the detectable fragment can be or consist of an agent that binds to the biomarker (e.g., an antibody or fragment thereof).

[00017] In some embodiments, the integration of the nucleic acid element does not substantially disrupt the expression of the polypeptide encoded at the target site (i.e., expression of the polypeptide at the target site continues substantially as it would if the subject were not receiving the integration gene therapy or the gene integration composition). In some embodiments, the integration occurs without the use of an exogenously supplied nuclease. In some embodiments, the integration occurs using one or more exogenously supplied nucleases.

[00018] According to various embodiments, the methods and compositions described herein are considered compatible with various gene therapy regimens. For example, in some embodiments, the subject receives a single dose of the gene therapy agent or gene integration composition. In some embodiments, the subject receives multiple doses of the gene therapy agent or gene integration composition (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more).

[00019] Furthermore, the methods and compositions described herein can be used at any of a plurality of time points after the gene therapy treatment (e.g., several hours, days, weeks, or months after the subject has received the gene therapy). Accordingly, in some embodiments, the detecting step is performed 1, 2, 3, 4, 5, 6, 7, 8, or more weeks after the subject has received the gene therapy treatment or the gene integration composition. In some embodiments, the detecting step is performed at multiple time points after the subject has received the gene therapy treatment or the gene integration composition. In some embodiments, the detecting step is performed (e.g., multiple times) at least 3 months after the subject has received the gene therapy treatment or the gene integration composition.

[00020] Surprisingly, it has been discovered that some embodiments of the invention are able to provide a benefit (e.g., facilitate monitoring and/or adjustment of therapy) to a subject at various stages of life when receiving gene therapy, and, in some embodiments, the disclosed methods can be used as a subject transitions from one stage of life to another. In some embodiments, the subject receives the gene therapy treatment or gene integration composition as an infant. In some embodiments, the subject receives the gene therapy treatment or gene integration composition before reaching adulthood (e.g., as a child). In some embodiments, the subject receives the gene therapy treatment or gene integration composition as an adult.

[00021] In particular, it is contemplated that the methods and compositions described herein are applicable to a variety of subjects, each of which potentially has confounding or complicating factors/conditions in addition to those that require the use of gene therapy. In addition, certain forms of gene therapy are known or suspected to potentially cause problematic reactions (e.g., autoimmune reactions, cytokine storms, etc.). Accordingly, in some embodiments, the present methods further comprise monitoring the subject for an autoimmune response to the gene therapy. In some embodiments, the present methods further comprise monitoring the subject for an abnormal cytokine response to the gene therapy (e.g., a cytokine storm).

[00022] The present disclosure also includes the recognition that gene therapy may need to be adjusted (e.g., enhanced or suppressed), and it is contemplated that various embodiments are advantageous for monitoring the need for and/or successful implementation of such adjustments. Accordingly, in some embodiments, the provided methods further comprise administering to the subject an additional treatment (e.g., an activating agent) if the level of the biomarker is lower than that which is indicative of achieving a therapeutically effective amount of the integrative gene therapy. Additionally or alternatively, in some embodiments, the provided methods further comprise delivering to the subject an additional treatment (e.g., a deactivating agent) that reduces or inhibits the expression of the payload delivered by the gene therapy treatment agent if the level of the biomarker exceeds a level that is indicative of an optimal or safe level of the payload.

[00023] In this application, the terms "about" and "approximately" are used interchangeably. Any references to publications, patents or patent applications cited herein are incorporated herein by reference in their entirety. Any numbers used in this application, with or without "about"/"approximately", are intended to cover any normal variations appreciated by a person of ordinary skill in the relevant technical field.

[00024] Other features, objects and advantages of the present invention will be apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only and not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.

Brief description of graphic materials

[00025] FIG. 1 shows a schematic of the GeneRide™ t (AAV) construct before and after homologous recombination (HR) mediated integration into the genome at the target albumin locus Alb. Expression from the GeneRide™ edited Alb locus can result in the simultaneous production of albumin-2A and the transgene as separate proteins.

[00026] FIG. 2 depicts exemplary methods for analyzing genomic DNA (gDNA) integration. As shown, such methods can be applied to an analysis of gDNA edited with GeneRide™ at the albumin (Alb) locus. In step 1 depicted, a product from isolated gDNA is amplified using long-range PCR (LR-PCR) with primers F1/R1. In step 2 depicted, the purified product from step 1 is amplified with primers F2/R2 in a nested qPCR.

[00027] FIG. 3 shows an exemplary approach for quantifying episomal DNA. As shown, the number of episomal copies can be determined by qPCR using a standard curve generated using a linearized episomal plasmid.

[00028] FIG. 4 shows an exemplary approach to analyzing mRNA comprising a nucleic acid sequence encoding the 2A peptide. The copy number of the fusion mRNAs is determined using droplet digital PCR (ddPCR) with the Fwd/R _F primer set. The copy number of endogenous Alb is measured using ddPCR with the Fwd/R _E primer set and used for normalization.

[00029] FIG. 5A-FIG. 5B represent an exemplary approach for detecting and quantifying polypeptides in blood plasma. For example, albumin-2A in blood plasma can be analyzed using the illustrated methods. FIG. 5A) depicts a sandwich ELISA comprising a capture antibody and a detection antibody. The illustration depicts an anti-2A antibody for capturing albumin-2A and a labeled anti-albumin antibody for detection. Capture and detection antibodies specific for other polypeptides in plasma can be used to detect other polypeptides, such as albumin, human factor IX, and cyno A1AT. FIG. 5B) depicts standard curves based on recombinant mouse albumin-2A in PBST buffer or 10% mouse serum.

[00030] FIG. 6A-FIG. 6C show in vivo detection and analysis of episomal DNA. Neonatal (p2) mice were injected with 1e14 gv/kg (vector genomes per kilogram) of hF9-DJ. FIG. 6A) The number of episomal copies decreased exponentially with time after injection. FIG. 6B) The injection did not significantly affect liver growth in the animals. FIG. 6C) The injection did not significantly affect body weight gain in the animals.

[00031] FIG. 7A-FIG. 7D show in vivo detection, monitoring, and analysis of biomarker 2A over time. Neonatal C57 mice at p2 were injected intravenously with 1e14 gv/kg hF9-DJ and tissues were collected for analysis at 1, 2, 3, 4, and 8 weeks post-injection (n=5/group). FIG. 7A) Genomic DNA integration of biomarker 2A in the liver was assessed using long-stranded PCR/qPCR and expressed as a percentage of endogenous Alb. FIG. 7B-FIG. 7C) ALB-2A and total mouse albumin in plasma were measured by ELISA. FIG. 7D) Correlation of the data shown in FIG. 7B and FIG. 7C. Analysis of the ratio of peptide-2A-labeled albumin to total plasma albumin confirms that the observed increase in plasma ALB-2A levels is associated with an exponential increase in endogenous albumin after birth.

[00032] FIG. 8A-FIG. 8B demonstrate in vivo detection, monitoring, and analysis of biomarker-delivered payload over time. Neonatal C57 mice at p2 were intravenously injected with 1e14 gv/kg hF9-DJ and tissues were collected for analysis at 1, 2, 3, 4, and 8 weeks post-injection. FIG. 8A) Human factor IX was quantified in mouse plasma using a human factor IX-specific ELISA. FIG. 8B) Correlation of the data shown in FIG. 7B and FIG. 8A.

[00033] FIGS. 9A-9E show the detection and analysis of biomarker and payload delivery at two doses and two age groups. Neonatal (p2) or juvenile (p21) C57 mice were injected intravenously with 1e13 or 1e14 gv/kg hF9-DJ and tissues were collected for analysis at 8 weeks post-injection (n=6-8/group). Animals dosed at p2 were 8 weeks old and animals dosed at p21 were 11 weeks old at the time of collection for analysis. FIG. 9A) Plasma ALB-2A measured by ELISA. FIG. 9B) Plasma human factor IX measured by ELISA. FIG. 9C) Fusion mRNA in liver quantified by ddPCR and expressed as a percentage of endogenous albumin mRNA. FIG. 9D) Genomic DNA integration in liver was assessed using long-fragment PCR/qPCR and expressed as a percentage of endogenous Alb. FIG. 9E) Episomal copies per cell measured in liver by qPCR.

[00034] FIG. 10A-FIG. 10C show in vivo detection, monitoring, and analysis of biomarker-delivered payload in subjects of different age groups. Neonatal FvB/NJ mice (p2), juvenile mice (p21), or adult mice (p42 and p63) were injected intravenously with 1e14 gv/kg hF9-DJ and tissues were collected for analysis at 4 weeks post-injection (n=6-9/group). At the time of collection for analysis, animals dosed at p2 were 4 weeks old, those dosed at p21 were 7 weeks old, those dosed at p42 were 11 weeks old, and those dosed at p63 were 14 weeks old. FIG. 10A-FIG. 10B) Plasma ALB-2A and human factor IX were measured by ELISA. FIG. 10C) The linear regression of plasma ALB-2A versus human factor IX is R ² = 0.93.

[00035] FIG. 11A-FIG. 11C show in vivo detection, monitoring, and analysis of biomarker-delivered payloads from viral vectors containing different homology arms for genomic integration. Neonatal FvB/NJ (p2) mice were injected intravenously with A1AT-DJ at a final dose of 1e13 or 1e14 gv/kg. HA-750bp corresponds to the 750bp transgene with homology arms, and HA-1kbp corresponds to the transgene with 1 kb homology arms. Animals were collected for analysis at 6 weeks post-injection (n=6-9/group). FIG. 11A-FIG. 11B) Plasma ALB-2A and cyno A1AT were measured by ELISA. FIG. 11C) The linear regression of ALB-2A versus A1AT is ^R2 = 0.91.

[00036] FIG. 12A-FIG. 12B show in vivo detection, monitoring, and analysis of cellular internal payload delivered with a biomarker over time. Neonatal Mut ^−/− ;Tg ^INS-MCK-Mut (p2) mice were injected intravenously with various doses of DJ-hMUT (1e13, 3e13, or 1e14 vg/kg) and harvested for analysis at 3 months. FIG. 12A) Integration of genomic DNA into the liver was assessed using long-stranded PCR/qPCR and expressed as a percentage of endogenous Alb. ALB-2A in plasma was measured by ELISA. FIG. 12B) Protein expression of the integrated transgene, human MUT, was measured by Western blot in liver lysates of MCK-MUT mice. Circulating ALB-2A was found to be linearly correlated with levels of genomic integration in the liver, as well as with MUT protein levels.

[00037] FIG. 13A-FIG. 13B demonstrate that payload expression can be increased following a single administration (i.e., selective expansion of GeneRide™ edited hepatocytes) and that the increase in payload levels can be monitored by analyzing biomarker levels. Neonatal Mut ^−/− ;Tg ^INS-MCK-Mut (p2) mice were injected intravenously with 1e14 gv/kg DJ-hMUT and collected for analysis for 7 months. FIG. 13A-13B) Human MUT protein expressed from the integrated transgene in MUT ^−/− mice was analyzed in liver lysates by Western blotting using β-actin as a loading control. ALB-2A (blotted for 2A) and total albumin were also analyzed in these liver lysates. Vehicle-treated MUT ^+/- mice and wild-type B6 mice served as controls. Note: The MUT protein in MUT ^-/- hepatocytes expressed from the transgene is human, whereas the endogenous protein in MUT ^+/- mice and wild-type B6 mice is murine.

[00038] FIG. 14 shows standard curves based on recombinant mouse ALB-2A prepared in sample diluent only or 1% mouse plasma using an optimized ELISA method for ALB-2A.

[00039] FIGS. 15A-15B show in vivo detection, monitoring, and analysis of biomarker-delivered payload in wild-type mice and a NAFLD (DIO) mouse model. Adult mice (~9 weeks old) were injected intravenously with 1e14 gv/kg hF9-DJ, and plasma samples were collected at week 1 and then biweekly for 16 weeks. ALB-2A and human factor IX were quantified in mouse plasma by ELISA.

[00040] FIGS. 16A-16C show the detection and analysis of biomarker and payload delivery in a dose-dependent manner. Neonatal FvB(p2) mice were injected intravenously with 4.1e12, 1.2e13, 3.7e13, 1.1e14, 3.3e14, and 1e15 gv/kg chA1AT-DJ and tissues were collected for analysis at 4 weeks post-injection (n= 5/group). FIGS. 16A-B) show plasma ALB-2A and cA1AT levels measured by ELISA, respectively. FIG. 16C) shows that the linear regression of ALB-2A versus A1AT is ^R2 =0.97.

[00041] FIG. 17A-C show the detection and analysis of gDNA integration and payload delivery in a dose-dependent manner. Neonatal Mut ^+/- ; Tg ^INS-MCK-Mut mice (p0) were injected intravenously with low, medium, and high doses of DJ-mMUT (2.1e13, 6.7e13, or 2.0e14 gv/kg) and collected for analysis at 90 days post-dosing. FIG. 17A shows plasma ALB-2A levels measured by ELISA (n=18 low dose, n=19 medium dose, n=19 high dose). FIG. 17B shows the percentage of gDNA integration in the liver (n=26 low dose, n=25 medium dose, n=28 high dose). FIG. 17C shows the correlation of ALB-2A and gDNA integration percentage for animals with both assays.

Definitions

[00042] In order to make the present invention more understandable, definitions of certain terms are provided below. Additional definitions of the following terms and other terms are provided throughout the specification.

[00043] About: The term "about," when used herein in relation to a value, refers to a value that is similar in context to the value being referenced. In general, those skilled in the art, familiar with the context, will appreciate the appropriate degree of deviation encompassed by the word "about" in this context. For example, in some embodiments, the term "about" may encompass a range of values that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated value.

[00044] Activating agent: As used herein, the term "activating agent" refers to an agent whose presence or level correlates with an increased level or activity of a target compared to that observed in the absence of the agent (or with the agent at a different level). In some embodiments, an activating agent is an agent whose presence or level correlates with a target level or activity that is comparable to or greater than a specified reference level or activity (e.g., observed under appropriate reference conditions, such as the presence of a known activating agent, e.g., a positive control). In some embodiments, an activating agent binds to or is otherwise associated with an activating element to exert its effect.

[00045] Adult: As used herein, the term "adult" refers to a human being eighteen years of age or older. In some embodiments, an adult human being has a weight in the range of about 90 pounds to about 250 pounds.

[00046] Associated: Two events or entities are "associated" with each other, as that term is used herein, if the presence, level, and/or form of one correlates with the presence, level, and/or form of the other. For example, a particular entity (e.g., a polypeptide, genetic signature, metabolite, microbe, etc.) is said to be associated with a particular disease, disorder, or condition if its presence, level, and/or form correlates with the incidence of and/or susceptibility to the disease, disorder, or condition (e.g., in a relevant population). In some embodiments, two or more entities are physically "associated" with each other if they interact, directly or indirectly, such that they are and/or remain in physical proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently associated with each other; In some embodiments, two or more entities physically associated with each other are not covalently associated with each other, but are non-covalently associated, such as through hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic properties, and combinations thereof.

[00047] Biological Sample: As used herein, the term "biological sample" generally refers to a sample obtained from or derived from a biological source (e.g., tissue, organism, or cell culture) of interest as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample is or includes biological tissue or fluid. In some embodiments, the biological sample can be or include bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; body fluids containing cells; free-floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; stool; lymph; gynecologic fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages, such as ductal lavage or bronchoalveolar lavage; aspirates; scrapings; bone marrow samples; tissue biopsy samples; surgical samples; feces, other body fluids, secretions and/or excrements; and/or cells therefrom, etc. In some embodiments, the biological sample is or includes cells obtained from a human. In some embodiments, the cells obtained are or include cells from the individual from whom the sample was obtained. In some embodiments, the sample is a "primary sample" obtained directly from a source of interest by any suitable method. For example, in some embodiments, the primary biological sample is obtained by methods selected from the group consisting of a biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of a body fluid (e.g., blood, lymph, stool, etc.), etc. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation that is obtained by processing (e.g., by removing one or more components and/or adding one or more agents) a primary sample. For example, by filtration using a semipermeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins isolated from the sample or obtained by subjecting the primary sample to methods such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.

[00048] Biomarker: The term "biomarker" is used herein consistent with its use in the art to refer to a compound whose presence, level, or form correlates with a particular biological event or condition of interest, such that it is considered a "marker" of that event or condition. Among other things, the present disclosure provides biomarkers for gene therapy (e.g., which are useful for assessing one or more features or characteristics of a gene therapy treatment, such as, for example, the extent, level, and/or persistence of payload expression). In some embodiments, the biomarker is a cell surface marker. In some embodiments, the biomarker is intracellular. In some embodiments, the biomarker is extracellular (e.g., secreted or otherwise produced or present outside of cells, such as in a body fluid such as blood, urine, tears, saliva, cerebrospinal fluid, etc.). In some embodiments, the present disclosure demonstrates the effectiveness of biomarkers that can be detected in a sample obtained from a subject who has received a gene therapy for use in assessing one or more properties or characteristics of the gene therapy; in some such embodiments, the sample is cells, tissue, and/or fluid other than those into which the gene therapy has been delivered and/or those in which the payload is active.

[00049] Detectable fragment: As used herein, the term "detectable fragment" refers to any entity (e.g., a molecule, complex, or part or component thereof). In some embodiments, a detectable fragment is provided and/or used as a separate molecular entity; in some embodiments, it is part of and/or associated with another molecular entity. Examples of detectable fragments include, but are not limited to: various ligands. radionuclides (e.g. ³ H, ¹⁴ C, ¹⁸ F, ¹⁹ F, ³² P, ³⁵ S, ¹³⁵ I, ¹²⁵ I, ¹²³ I, ⁶⁴ Cu, ¹⁸⁷ Re, ¹¹¹ In, ⁹⁰ Y, ^99m Tc, ¹⁷⁷ Lu, ⁸⁹ Zr, etc.), fluorescent dyes (see below for specific examples of fluorescent dyes), chemiluminescent agents (such as acridine esters, stabilized dioxetanes, etc.), bioluminescent agents, spectrally resolved inorganic fluorescent semiconductor nanocrystals (i.e. quantum dots), metal nanoparticles (e.g. gold, silver, copper, platinum, etc.), nanoclusters, paramagnetic metal ions, enzymes (see below for specific examples of enzymes), colorimetric labels (such as dyes, colloidal gold, etc.), biotin, dioxygenin, haptens, antibodies and/or proteins for which antisera or monoclonal antibodies are available.

[00050] Child: As used herein, the term "child" refers to a person between the ages of two and 18 years. Body weight can vary widely at different ages and among different children, with a typical range being 30 to 150 pounds.

[00051] Combination Therapy: As used herein, the term "combination therapy" refers to situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents, such as a gene therapy and a non-gene therapy mode of action). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered before any doses of the second regimen are administered); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administering" a combination therapy may involve administering one or more agent(s) or mode(s) of action to a subject receiving the other agent(s) or mode(s) of action in the combination. For clarity, a combination therapy does not require that the individual agents be administered together in a single composition (or even necessarily at the same time).

[00052] Composition: Those skilled in the art will appreciate that the term "composition" as used herein may be used to refer to a discrete physical entity that includes one or more of the specified components. In general, unless otherwise specified, the composition may be in any form - for example, a gas, gel, liquid, solid, etc.

[00053] Inactivating Agent: As used herein, the term "inactivating agent" refers to an agent whose presence or level correlates with a reduced level or activity of a target compared to that observed in the absence of the agent (or with the agent at a different level). In some embodiments, an inactivating agent is an agent whose presence or level correlates with a target level or activity that is comparable to or lower than a certain reference level or activity (e.g., observed under appropriate reference conditions, such as the presence of a known activating agent, e.g., a positive control). In some embodiments, the inactivating agent binds to or is otherwise associated with a inactivating element to exert its effect.

[00054] Determine: Many of the methodologies described herein include a "determining" step. Those skilled in the art, upon reading this specification, will understand that such "determining" can be used or performed using any of a variety of methods available to those skilled in the art, including, for example, the specific methods expressly set forth herein. In some embodiments, determining includes manipulating a physical sample. In some embodiments, determining includes viewing and/or manipulating data or information, such as by using a computer or other processing device adapted to perform the appropriate analysis. In some embodiments, determining includes obtaining relevant information and/or materials from a source. In some embodiments, determining includes comparing one or more characteristics of a sample or object to a comparable standard.

[00055] Gene: As used herein, the term "gene" refers to a DNA sequence that encodes a gene product (e.g., an RNA product and/or a polypeptide product). In some embodiments, a gene includes a coding sequence (e.g., a sequence that encodes a particular gene product); in some embodiments, a gene includes a non-coding sequence. In some specific embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences. In some embodiments, a gene may include one or more regulatory elements (e.g., promoters, enhancers, silencers, termination signals) that, for example, can control or influence one or more aspects of gene expression (e.g., cell type-specific expression, inducible expression). In some embodiments, a gene is located or is found (or has a nucleotide sequence identical to that located or found) in a genome (e.g., in or on a chromosome or other replicating nucleic acid).

[00056] Gene Product or Expression Product: As used herein, the term "gene product" or "expression product" generally refers to RNA transcribed from a gene (before and/or after processing) or a polypeptide (before and/or after modification) encoded by RNA transcribed from a gene.

[00057] "Improve," "increase," "inhibit," or "decrease": As used herein, the terms "improve," "increase," "inhibit," "decrease," or grammatical equivalents thereof, refer to values that are relative to a baseline or other reference measurement. In some embodiments, a suitable reference measurement may be or include a measurement in a particular system (e.g., in a single individual) under comparable conditions without the presence of (e.g., before and/or after) a particular agent or treatment or in the presence of a corresponding comparable reference agent. In some embodiments, a suitable reference measurement may be or include a measurement in a comparison system that is known or expected to respond in a particular manner in the presence of a corresponding agent or treatment.

[00058] Infant: :As used herein, the term "infant" refers to a human being under two years of age. The typical body weight of an infant is between 3 pounds and 20 pounds.

[00059] Nucleic acid: As used herein, refers in the broadest sense to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As will be apparent from the context, in some embodiments, a "nucleic acid" refers to an individual nucleic acid unit (e.g., a nucleotide and/or nucleoside); in some embodiments, a "nucleic acid" refers to an oligonucleotide chain that includes individual nucleic acid units. In some embodiments, a "nucleic acid" is or includes RNA; in some embodiments, a "nucleic acid" is or includes DNA. In some embodiments, a nucleic acid is, includes, or consists of one or more naturally occurring nucleic acid units. In some embodiments, the nucleic acid is, includes, or consists of one or more nucleic acid analogs. In some embodiments, the nucleic acid analog differs from the nucleic acid in that it does not use a phosphodiester backbone. In some embodiments, the nucleic acid is, includes, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, the nucleic acid is, includes, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3'-methyl adenosine, 5-methylcytidine, C-5-propynyl-cytidine, C-5-propynyl-uridine, 2-aminoadenosine, C5-bromuridine, C5-fluorouridine, C5-ioduridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deaadenosine, 7-deaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as RNA or protein. In some embodiments, the nucleic acid includes one or more introns. In some embodiments, nucleic acids are produced by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), propagation in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues. In some embodiments, the nucleic acid is partially or completely single-stranded; in some embodiments, the nucleic acid is partially or completely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element that encodes or is complementary to a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[00060] Peptide: As used herein, the term "peptide" or "polypeptide" refers to any polymeric chain of amino acids. In some embodiments, a peptide has an amino acid sequence that is naturally occurring. In some embodiments, a peptide has an amino acid sequence that is not naturally occurring. In some embodiments, a peptide has an amino acid sequence that is engineered, i.e., designed and/or produced by human hands. In some embodiments, a peptide may be composed of naturally occurring amino acids, unnatural amino acids, or both. In some embodiments, a peptide may be composed of only naturally occurring amino acids or only unnatural amino acids. In some embodiments, a peptide may include D-amino acids, L-amino acids, or both. In some embodiments, a peptide may include only D-amino acids. In some embodiments, a peptide may include only L-amino acids. In some embodiments, a peptide is linear. In some embodiments, the term "peptide" may be appended to the name of a reference peptide, activity, or structure; in such cases, it is used herein to refer to peptides that have the corresponding activity or structure and thus can be considered to be members of the same class or family of peptides. For each such class, exemplary peptides within the class, the amino acid sequences of which and/or functions are known, are provided in this specification and/or will be known to those skilled in the art; in some embodiments, such exemplary peptides are reference peptides for the class or family of peptides. In some embodiments, a member of a class or family of peptides exhibits significant sequence homology or identity, shares a common sequence motif (e.g., a signature sequence element), and/or shares a common activity (in some embodiments, at a comparable level or within a designated range) with the reference peptide of the class; in some embodiments, with all peptides of the class). For example, in some embodiments, the member peptide exhibits an overall degree of homology or sequence identity to a reference peptide that is at least about 30-40%, and often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and/or includes at least one region (e.g., a conserved region, which in some embodiments may be or consist of a signature sequence element) that exhibits very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98% or 99%. Such a conserved region typically spans at least 3-4 and often up to 20 or more amino acids; in some embodiments, the conserved region spans at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.

[00061] Polypeptide: As used herein, the term "polypeptide" or "protein" refers to a polymer of at least three amino acid residues. In some embodiments, the polypeptide includes one or more, or all, naturally occurring amino acids. In some embodiments, the polypeptide includes one or more, or all, non-naturally occurring amino acids. In some embodiments, the polypeptide includes one or more, or all, D-amino acids. In some embodiments, the polypeptide includes one or more, or all, L-amino acids. In some embodiments, the polypeptide includes one or more side groups or other modifications, such as modifying or attached to one or more amino acid side chains, at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or any combination thereof. In some embodiments, the polypeptide includes one or more modifications such as acetylation, amidation, aminoethylation, biotinylation, carbamylation, carbonylation, citrullination, deamidation, deelimination, elimination, glycosylation, lipidation, methylation, pegylation, phosphorylation, sumoylation, or combinations thereof. In some embodiments, the polypeptide can participate in one or more intra- or intermolecular disulfide bonds. In some embodiments, the polypeptide can be cyclic and/or can include a cyclic portion. In some embodiments, the polypeptide can be non-cyclic and/or does not include a cyclic portion. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide can include a cross-linked polypeptide. In some embodiments, the polypeptide participates in the formation of a non-covalent complex by non-covalent or covalent association with one or more other polypeptides (e.g., as in an antibody). In some embodiments, the polypeptide has an amino acid sequence that occurs in nature. In some embodiments, the polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, the polypeptide has an amino acid sequence that is engineered, i.e., it is designed and/or produced by human hands. In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity, or structure; in such cases, it is used herein to refer to polypeptides that have the corresponding activity or structure and thus can be considered as members of the same class or family of polypeptides. For each such class, exemplary polypeptides within the class whose amino acid sequences and/or functions are known are provided in this specification and/or will be known to those skilled in the art; in some embodiments, such exemplary polypeptides are reference polypeptides for the class or family of polypeptides. In some embodiments, a member of a class or family of polypeptides exhibits significant sequence homology or identity, shares a common sequence motif (e.g., a signature sequence element), and/or shares a common activity (in some embodiments, at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments, with all polypeptides of the class). For example, in some embodiments, the member polypeptide exhibits an overall degree of homology or sequence identity to a reference polypeptide that is at least about 30-40%, and often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and/or includes at least one region (e.g., a conserved region, which in some embodiments may be or consist of a signature sequence element) that exhibits very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98% or 99%. Such a conserved region typically spans at least 3-4 and often up to 20 or more amino acids; in some embodiments, the conserved region spans at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, the useful polypeptide may include a fragment of a parent polypeptide. In some embodiments, the useful polypeptide may include a plurality of fragments, each of which is in a different spatial arrangement relative to one another in the same parent polypeptide than in the polypeptide of interest (e.g., fragments that are directly linked in the parent polypeptide may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), such that the polypeptide of interest is derived from its parent polypeptide.

[00062] Subject: As used herein, the term "subject" means an organism, typically a mammal (e.g., a human, in some embodiments, including prenatal human forms). In some embodiments, the subject suffers from a relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is a human with one or more characteristics that characterize a predisposition to, or is at risk for, the disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is a human being diagnosed and/or treated.

[00063] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting a full or nearly full degree or level of a characteristic or property of interest. A person of ordinary skill in biology will recognize that biological and chemical phenomena rarely, if ever, reach completion and/or proceed to completeness, or achieve or avoid an absolute outcome. Therefore, the term "substantially" is used herein to reflect the potential lack of completeness inherent in many biological and chemical phenomena.

Detailed description of specific implementation options

[00064] The present disclosure provides technologies for monitoring and/or otherwise evaluating gene therapy. As described herein, the present disclosure relates to detecting and evaluating a biomarker(s) that are generated as a result of gene integration therapy treatment, the presence and relative amounts of which reveal information about the payload delivered by the gene therapy treatment, such as information about the presence, amount, and/or kinetics of the delivered payload. In one aspect of the present disclosure, the presence, amount, and/or kinetics of the biomarker acts as a surrogate for determining the presence, amount, and/or kinetics of the delivered payload. In some embodiments of the disclosure, the biomarker is evaluated from a biological sample taken from a subject that has received integration gene therapy treatment. In some embodiments of the disclosure, the biomarker may be evaluated in a non-tissue biological sample taken from a subject. In some embodiments of the disclosure, the payload is delivered (e.g., via delivery of a corresponding transgene) and remains in the tissue of a subject treated with the integration gene therapy.

Integration gene therapy

[00065] Gene therapy introduces genetic material into the cells of a subject, typically to express a payload that can compensate for an abnormal gene or otherwise provide a beneficial effect for the subject. Integration gene therapy introduces genetic material that integrates into a genetic sequence (i.e., a target site) present in a recipient cell.

[00066] Those skilled in the art are familiar with a variety of technologies for integrating genetic material into a target site of interest in a recipient cell or organism. Such integrated genetic elements may include a nucleic acid sequence (i.e., a "transgene," as that term is used herein) that encodes a payload to be delivered to the host cell or organism. Typically, the transgene is delivered in the context of a vector; those skilled in the art are familiar with viral and non-viral vector systems that can be successfully used to achieve transgene integration.

[00067] The present disclosure provides for identification of the source of the problem using various integration gene therapy technologies.

[00068] For example, the present disclosure recognizes that low-throughput or inefficient integration can limit the utility of gene therapy strategies. If a vector does not integrate, it is typically lost as cells divide during tissue growth or regeneration, and any benefits that the delivered transgene (or payload) provides or could provide will also be lost. A similar difficulty arises even when integration is initially successful but is subsequently lost, such as by recombination or death of the recipient cell.

[00069] The present disclosure also notes that many gene integration technologies or events cannot or cannot precisely control the target integration site, and that the integration site can significantly affect the extent and/or timing of transgene expression and/or can affect the health or even viability of the host cell. Furthermore, the present disclosure recognizes that even some "targeted" gene integration technologies can be negatively affected by the integration site, such that transgene expression may not be able to be achieved and/or maintained at the desired level and/or for the desired period of time.

[00070] The present disclosure also recognizes that many gene therapy approaches introduce a transgene in operative association with a promoter (e.g., an exogenous promoter), and that the expression characteristics of such a promoter can negatively impact recipient cells, including potentially increasing the risk of uncontrolled proliferation (e.g., cancer), particularly for promoters that provide high levels of gene expression.

[00071] Thus, the present disclosure makes it clear that one of the problems with many integrative gene therapy treatments is the inability or impossibility to monitor the expression of the corresponding payload, especially over time. Given that many payloads are or may be intracellular and/or the tissues in which they are to be expressed and/or active may be relatively inaccessible, regular monitoring is often not performed.

[00072] The present disclosure contributes to the discovery that certain gene integration technologies can generate an effective biomarker for successful transgene integration and payload expression. Moreover, the present disclosure demonstrates that some such technologies generate a biomarker that can be assessed from readily available biological samples (e.g., blood, urine, tears, etc.). Thus, the present disclosure provides technologies that improve integration gene therapy, in particular by providing systems for monitoring (e.g., detecting and/or quantifying, in many embodiments at multiple time points) a biomarker generated as a result of successful integration and reflecting payload expression.

[00073] It is contemplated that any of a variety of integrative gene therapy technologies may be used. As a non-limiting example, in some embodiments, integrative gene therapy may be or include the use of a vector-based system (e.g., a viral vector-based system), a non-viral vector-based system, a nuclease-mediated system, and/or the use of the GENERIDE™ system, or any combination thereof.

Vector Based Systems

[00074] In some embodiments, the integration gene therapy may be or consist of a vector-based system (e.g., a viral vector). Typically, a vector-based system includes a virus or viral genetic material into which a fragment of foreign DNA can be inserted for transfer into a cell. Any virus that includes a DNA step in its life cycle can be used as a viral vector within the scope of some embodiments of the present disclosure. As a non-limiting example, the viral vector-based system can be a single-stranded DNA virus, a double-stranded DNA virus, an RNA virus that has a DNA step in its life cycle, such as retroviruses. In some embodiments, the viral vector can be delivered via a pharmaceutically acceptable formulation, such as a liposome or a lipid particle (e.g., a micro- or nanoparticle).

[00075] As one non-limiting example, one virus of interest is adeno-associated virus. By adeno-associated virus, or "AAV", is meant the virus itself or derivatives thereof. The term encompasses all subtypes and both naturally occurring and recombinant forms unless otherwise specified, e.g., AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10), AAV type 11 (AAV-11), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, ovine AAV, hybrid AAV (e.g., an AAV comprising the capsid protein of one AAV subtype and the genomic material of another subtype), AAV comprising a mutant AAV capsid protein or a chimeric AAV capsid protein (i.e., a capsid protein with regions, domains, or individual amino acids that are derived from two or more different AAV serotypes, e.g., AAV-DJ, AAV-LK3, AAV-LK19). "Primate AAV" refers to AAVs that infect primates, "non-primate AAV" refers to AAVs that infect non-primates, "bovine AAV" refers to AAVs that infect mammals such as cattle, etc.

[00076] Regardless of the vector used (e.g., viral), to promote targeted integration, the targeted vector includes nucleic acid sequences that allow homologous recombination at the site of integration, such as sequences that allow homologous recombination with an albumin gene, a collagen gene, an actin gene, etc. This process requires nucleotide sequence homology, using a "donor" molecule, such as a targeted vector, to template repair a "target" molecule, i.e., a nucleic acid into which the nucleic acid sequence is integrated, such as a target locus in a cellular genome, and results in the transfer of genetic information from the donor to the target. Thus, in the target vectors of the compositions of the subject, the transgene to be integrated into the cellular genome can be flanked by sequences that contain sufficient homology to the genomic sequence at the cleavage site, such as 70%, 80%, 85%, 90%, 95% or 100% homology with nucleotide sequences flanking the cleavage site, such as within about 50 bases or less from the cleavage site, such as within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases or immediately flanking the target integration site, to support homologous recombination between it and the genomic sequence to which it has homology. Approximately 25, 50, 100, 250, or 500 nucleotides or more of sequence homology between the donor and the genomic sequence will support homologous recombination between them.

Non-viral vector systems

[00077] In some embodiments, the integration gene therapy may be or consist of a non-viral vector system. In some embodiments, the non-viral vector system may be or consist of a plasmid, a polymer-based particle, ccDNA (closed-end DNA), liposomes, minicircles, and combinations thereof. In some embodiments, the non-viral vector system may be or include the use of chemical carriers, electroporation, the use of ballistic DNA (e.g., particle bombardment), sonoporation, photoporation, magnetofection, hydroporation, and any combination thereof.

[00078] Similar to the above description, regardless of the type(s) of non-viral vector system(s) used, to facilitate targeted integration, it is necessary to use/deliver to the target site one or more nucleic acid sequences that favor homologous recombination at the integration site.

Nuclease-mediated integration

[00079] According to various embodiments, nuclease-mediated integration uses one or more nucleases, enzymes that were developed or originally identified in bacteria that cut DNA. Typically, nuclease-mediated integration is a two-step process. First, an exogenous nuclease capable of cutting one or both strands in double-stranded DNA is directed to the desired site of the synthetic guide RNA and makes a specific cut. After the nuclease makes the desired cut or cuts, the cell's DNA repair machinery is activated and completes the editing process via NHEJ (non-homologous end joining) or, less commonly, HDR (homologous repair).

[00080] In some embodiments, NHEJ can occur in the absence of a DNA template for the cell to copy when repairing a DNA cut. This is the primary or default pathway used by the cell to repair double-strand breaks. The NHEJ mechanism can be used to introduce small insertions or deletions known as indels, which results in gene dysfunction. NHEJ creates insertions and deletions in DNA through its repair mechanism, and can also result in off-target, unwanted mutations, including chromosomal aberrations.

[00081] Nuclease-mediated HDR occurs when a nuclease, a guide RNA, and a DNA template that resembles the cut DNA are co-delivered. The cell can then use this template to construct repair DNA, resulting in the replacement of defective genetic sequences with correct ones. In some embodiments, the HDR mechanism is the preferred repair pathway when using a nuclease-based approach to insert a corrective sequence due to its high fidelity. However, most genome repair after nuclease cuts continues to use the NHEJ mechanism. The more frequent NHEJ repair pathway has the potential to cause unwanted mutations at the cut site, limiting the range of diseases that can currently be addressed by any nuclease-mediated integration approaches.

GeneRide™ Technology Platform

[00082] GeneRide™ is a genome editing technology that utilizes homologous recombination, or HR, a natural DNA repair process that maintains genomic fidelity. By utilizing HR, GeneRide™ allows polynucleotides to be inserted into specific target genomic regions without the use of exogenous nucleases. The targeted integration of GeneRide™ polynucleotides is designed to utilize endogenous promoters at these target regions to provide high levels of tissue-specific gene expression without the deleterious problems associated with the use of exogenous promoters. In some embodiments of the present disclosure, GeneRide™ is used to deliver a polynucleotide encoding a payload into a host cell or organism.

[00083] GeneRide™ technology can be used to precisely integrate a polynucleotide encoding a therapeutic payload into a patient's genome to provide a stable therapeutic effect. Because GeneRide™ is designed to have such a long-lasting therapeutic effect, it can be used to treat target disorders in pediatric patients where it is critical to provide treatment early in the patient's life before irreversible pathological processes occur.

[00084] In some embodiments, GeneRide™ uses an AAV vector to deliver a gene into the cell nucleus. It then uses homologous recombination to stably integrate the corrective gene into the recipient genome at a location where it is regulated by an endogenous promoter, allowing for lifelong protein production even as the organism grows and changes over time.

[00085] GeneRide™ can provide precise, site-specific, stable, and durable integration of the correction gene into the host cell chromosome. In preclinical animal studies using GeneRide constructs, integration of the correction gene into a specific location in the genome has been observed.

[00086] The GeneRide™ modular system can be used to provide robust, tissue-specific gene expression that is reproducible for a variety of therapeutics delivered to one or more tissues. By replacing another transgene in the GeneRide™ construct, that transgene can be delivered to address a new therapeutic target while maintaining all other components of the construct. This approach will allow common manufacturing processes and assays to be used across multiple GeneRide™ product candidates and may shorten the development process for other treatment programs.

[00087] Previous work on non-damaging gene targeting is described in WO 2013/158309, incorporated herein by reference. Previous work on nuclease-free genome editing is described in WO 2015/143177, incorporated herein by reference.

Target site

[00088] Integration gene therapy for use in accordance with the present disclosure desirably achieves integration that achieves functional association of the integrated transgene with an active endogenous promoter such that transcription from the promoter generates a transcript that is propagated through the transgene. Moreover, in many embodiments, integration occurs at a target site selected such that such transcript includes an open reading frame different from the reading frame of the transgene.

[00089] In many embodiments, integration gene therapy for use in accordance with the present disclosure achieves integration at a target site in an endogenous gene (e.g., at a specific position within or adjacent to an endogenous gene) and spreads a transcript generated by transcription from the promoter of that gene at least to the extent that it is spread by the transgene.

[00090] In some embodiments, treatment with an integration gene therapy agent or a gene integration composition provides for the integration of a nucleic acid element comprising a sequence that encodes a payload into a target site in the genome of a subject. Those skilled in the art will appreciate that any of a variety of target sites may be suitable for use with the methods and compositions described herein. For example, in some embodiments, the target site encodes a polypeptide. In some embodiments, the target site may encode a polypeptide that is highly expressed in a subject (e.g., a subject that does not have a disease, disorder, or condition). In some embodiments, the integration of the nucleic acid element occurs at the 5' or 3' end of an endogenous gene that encodes the polypeptide. As a non-limiting example, in some embodiments, the target site encodes albumin.

[00091] It is contemplated that integrative delivery of genetic elements and/or transgenes can be accomplished for any tissue, including, but not limited to, the liver, central nervous system (e.g., spine), muscle, kidney, retina, and bone marrow hematopoietic cells.

Payloads

[00092] In accordance with various embodiments of the invention as described herein, any payload suitable for a particular application can be used. In accordance with various embodiments, the transgene encodes one or more payloads. As used herein, the terms "payload" and "gene of interest" (GFI) may be used interchangeably. In some embodiments, the payload is or consists of a peptide, a nucleic acid (e.g., an shRNA, a microRNA, and/or a nucleic acid encoding one or more peptides), and any combination thereof. In some embodiments, the integrative gene therapy treatments and/or gene integration compositions include a single payload. In some embodiments, the integrative gene therapy treatments and/or gene integration compositions include two or more payloads (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more).

[00093] For example, in some embodiments, the payload is or includes a peptide expressed intracellularly or a nucleic acid sequence encoding such a peptide (e.g., a transgene). As a non-limiting example, intracellularly expressed peptides include methylmalonyl-CoA mutase (MUT), phenylalanine hydroxylase (PAH), glucose-6-phosphatase catalytic subunit (G6PC), propionyl-CoA carboxylase, subunit alpha (PCCA), ATP-binding cassette transporter, subfamily B member 11 (ABCB11), ornithine carbamoyltransferase (OTC), UDP-glucuronosyltransferase, family 1, member A1 (UGT1A1), acid alpha-glucosidase (GAA), lysosomal acid glucosylceramidase (GBA), frataxin (FTX).

[00094] In some embodiments, the payload is or includes a peptide secreted extracellularly and/or a nucleic acid sequence encoding such a peptide (e.g., a transgene). As a non-limiting example, secreted peptides include human factor IX (F9) and alpha-1-antitrypsin (SERPINA1).

[00095] In some embodiments, the payload is a peptide that has intracellular or extracellular activity that promotes a biological process for treating a medical condition.

[00096] In some embodiments, the payload may be or consist of a peptide that is normally expressed in one or more healthy tissues, or a nucleic acid sequence encoding such a peptide. For example, in some embodiments, the payload is a peptide that is normally expressed in liver cells, or a nucleic acid sequence encoding such a peptide. For example, in some embodiments, the payload is a peptide that is normally expressed in muscle cells. For example, in some embodiments, the payload is a peptide that is normally expressed in cells of the central nervous system. For example, in some embodiments, the payload is a peptide that is normally expressed in cells of the eye.

[00097] In some embodiments, the payload may be or consist of a peptide that is not normally expressed in one or more healthy tissues (e.g., it is expressed ectopically), or a nucleic acid sequence encoding such a peptide. For example, in some embodiments, the payload is a peptide that is ectopically expressed in liver cells. For example, in some embodiments, the payload is a peptide that is normally expressed in muscle cells. For example, in some embodiments, the payload is a peptide that is normally expressed in cells of the central nervous system. For example, in some embodiments, the payload is a peptide that is normally expressed in cells of the eye.

[00098] In some embodiments, the payload includes an activating element (e.g., activated by an activating agent). In some embodiments, the payload includes a deactivating element (e.g., activated by a deactivating agent). In some embodiments, the activating or deactivating agent may be or consist of a small molecule,

Biomarkers

[00099] The present disclosure provides integration gene therapy technologies that generate a detectable biomarker that can act as a surrogate for payload expression.

[000100] According to the present disclosure, expression of an integrated transgene includes production of a transcript including at least one translatable open reading frame that encodes a polypeptide separate or separable from a payload encoded by the transgene. In some embodiments, translation of the transcript generates a single polypeptide that is cleaved to separate the payload from the biomarker; in some embodiments, translation of the transcript generates separate biomarker and payload polypeptides.

[000101] According to the present disclosure, the use of multiple biological samples is considered to be consistent with various embodiments. In some embodiments, the biomarker is or includes a detectable fragment that, upon translation of the polypeptide encoded by the target site, is fused to the polypeptide encoded by the target site. In some embodiments, the biomarker is or includes a detectable fragment that, upon translation of the polypeptide encoded by the target site, is fused to the polypeptide encoded by the payload. In some embodiments, binding of the biomarker to the payload may be advantageous, such as when the payload is a modified form of an endogenous protein and is therefore otherwise difficult or impossible to detect separately from the endogenous version. In some embodiments, the detectable fragment may be or consist of an agent that binds to the biomarker (e.g., an antibody or fragment thereof, such as an antibody that binds to the α2A peptide).

[000102] In some embodiments, the biomarker is or includes a 2A peptide. In some embodiments, the 2A peptide is selected from the group consisting of P2A, T2A, E2A, and F2A. In some embodiments, the biomarker can be or consist of a furin cleavage motif. As a non-limiting example, an array of furin cleavage motifs is described in Tian et al., FurinDB: A Databse of 20-Residue Furin Cleavage Site Motifs, Substrates and Their Associated Drugs, (2011), Int. J. Mol. Sci., vol. 12: 1060-1065. As a specific example, in some embodiments, the 2A peptide may have or include the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 1), and a transgene encoding such a 2A peptide may have or include the nucleotide sequence gccaccaacttcagcagcctgctgctgaaacaggccggcgacgtggaagagaaccctggccc (SEQ ID NO: 2). In some embodiments, the 2A peptide will have a sequence that is at least 80% identical to SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., at least 85%, 90%, 95%, 99% identical). In some embodiments, the 2A peptide or transgene encoding the 2A peptide may be or be generated as described in Kim et al., (2011) High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice, PLoS ONE, vol. 6(4):e18556; Wang et al. (2015) 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori, Sci Rep., vol. 5: 16273; Yu et al. (2012) Use of Mutated Self-Cleaving 2A Peptides as a Molecular Rheostat to Direct Simultaneous Formation of Membrane and Secreted Anti-HIV Immunoglobulins. PLoS ONE 7(11):e50438; or Trichas et al. (2008), Use of the viral 2A peptide for bicistronic expression in transgenic mice, BMC Biology, vol. 6:40.

[000103] In some embodiments, the biomarker may be or include a label (e.g., an immunological label), such as a myc, HA, FLAG, or other label. In some embodiments, the biomarker may be or consist of an internal ribosome entry site (IRES).

Detection of the detectable fragment

[000104] As described herein, detection (e.g., detection of a signal, such as a biomarker or a detectable fragment) in connection with the methods and compositions described herein can be achieved by any method suitable for use. For example, in some embodiments, the detection step is or includes an immunoassay or a nucleic acid amplification assay.

[000105] As described herein, many embodiments include the use of one or more biological samples (e.g., a fluid or tissue sample taken from a subject), and the method for detecting a biomarker may vary depending on the biological sample used in a particular embodiment. In accordance with the present disclosure, any of a plurality of biological samples is considered compatible with various embodiments. For example, in some embodiments, the biological sample is or includes hair, skin, stool, blood, plasma, serum, cerebrospinal fluid, urine, saliva, tears, vitreous humor, or mucus.

[000106] According to various embodiments, and depending on the particular biomarker(s) used, one of skill in the art will recognize one or more suitable methods for detecting or determining the presence and/or amount/level of a biomarker in a biological sample. In some embodiments, a biomarker may be detected using any of a variety of methods, including fluorescence, radioactivity, chemiluminescence, electrochemiluminescence, colorimetry, FRET, HTRF, isotopic techniques, binding partners (e.g., biotin/avidin, antibodies, hybridization), or any other known method for detecting a biomarker. In some embodiments, a biomarker may be detected by binding a detectable moiety (e.g., an exogenously added detectable molecule), such as an antibody comprising, for example, a label according to one or more of the above methods, or an enzyme (e.g., luciferase, β-gal).

Applications and additional aspects

[000107] Those skilled in the art will readily appreciate that the methods described herein may be useful in a variety of gene therapy applications. In some embodiments, the methods described herein may be useful for assessing whether a payload is over- or under-expressed compared to a desired or "normal" expression level. In some embodiments, the methods described herein may be used to predict or determine a potential adverse reaction to gene therapy (e.g., the development of a deleterious immune response, such as anti-drug antibodies and/or cytokine storms), potentially allowing for intervention and/or mitigation of the adverse reaction.

[000108] In particular, it is contemplated that the methods and compositions described herein are applicable to any of a variety of diseases, disorders, or conditions. As non-limiting examples, some embodiments may be useful for monitoring the course of therapy or other parameters of, among others, acidosis/acidemia (e.g., methylmalonic acidemia), urea cycle disorder, hemophilia, Crigler-Najjar disease, acute hepatic porphyria, hereditary ATTR amyloidosis, and/or alpha-1 antitrypsin deficiency (A1ATD).

[000109] According to various embodiments, the methods and compositions described herein are considered compatible with various gene therapy regimens. For example, in some embodiments, the subject receives a single dose of the gene therapy agent or gene integration composition. In some embodiments, the subject receives multiple doses of one or more gene therapy agents and/or gene integration compositions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more).

[000110] Furthermore, the methods and compositions described herein are contemplated to be applicable at any of a plurality of time points after the gene therapy(s) treatment(s) (e.g., several hours, days, weeks, or months after the subject has received the gene therapy). Accordingly, in some embodiments, the detecting step is performed 1, 2, 3, 4, 5, 6, 7, 8, or more weeks after the subject has received the gene therapy treatment or gene integration composition. In some embodiments, the detecting step is performed at multiple time points after the subject has received the gene therapy treatment or gene integration composition. In some embodiments, the detecting step is performed (e.g., multiple times) at least 3 months after the subject has received the gene therapy treatment or gene integration composition.

[000111] Surprisingly, it has been discovered that some embodiments of the invention are able to provide a benefit (e.g., facilitate monitoring and/or adjustment of therapy) to a subject at various stages of life when receiving gene therapy, and, in some embodiments, the disclosed methods can be used as a subject transitions from one stage of life to another. In some embodiments, the subject receives the gene therapy treatment or gene integration composition as an infant. In some embodiments, the subject receives the gene therapy treatment or gene integration composition before reaching adulthood (e.g., as a child). In some embodiments, the subject receives the gene therapy treatment or gene integration composition as an adult.

[000112] In particular, it is contemplated that the methods and compositions described herein are applicable to a variety of subjects, each of which potentially has confounding or complicating factors/conditions in addition to those that require the use of gene therapy. In addition, certain forms of gene therapy are known or suspected to potentially cause problematic reactions (e.g., autoimmune reactions, cytokine storms, etc.). Accordingly, in some embodiments, the present methods further comprise monitoring the subject for an autoimmune response to the gene therapy. In some embodiments, the present methods further comprise monitoring the subject for an abnormal cytokine response to the gene therapy (e.g., a cytokine storm).

[000113] The present disclosure also includes the recognition that gene therapy may need to be adjusted (e.g., enhanced or suppressed), and it is contemplated that various embodiments are advantageous for monitoring the need for and/or successful implementation of such adjustments. Accordingly, in some embodiments, the provided methods further comprise administering to the subject an additional treatment (e.g., an activating agent) if the level of the biomarker is lower than that which is indicative of achieving a therapeutically effective amount of the integrative gene therapy. Additionally or alternatively, in some embodiments, the provided methods further comprise delivering to the subject an additional treatment (e.g., a deactivating agent) that reduces or inhibits the expression of the payload delivered by the gene therapy treatment agent if the level of the biomarker exceeds a level that is indicative of an optimal or safe level of the payload.

Examples

Example 1: Examples of biomarker and payload detection methods

[000114] This example demonstrates exemplary methods that can be used to detect, monitor, and/or analyze a biomarker and/or payload in accordance with the present disclosure. Examples 2-7 utilized the methods and materials described herein.

Genomic DNA integration (INT) analysis: qPCR of long fragments

[000115] Genomic DNA (gDNA) was isolated from frozen mouse liver tissue using the Qiagen DNeasy kit. The long fragment PCR (LR-PCR) product amplified with primers F1/R1 (step 1) was purified with SPRI magnetic beads and used in a nested qPCR (step 2) with primer set F2/R2 (FIG. 2). Synthetic dsDNA including a fragment located upstream of the 5' homology arm of the gene of interest (GFI) (~2.1 kb) was used to generate a standard curve. Standards were run in parallel with samples. Tfrc was used as a loading control for normalization.

Episomal DNA analysis

[000116] Genomic DNA (gDNA) was isolated from mouse liver using the Qiagen DNeasy kit. Episomal copy number was determined by qPCR (FIG. 3) using a standard curve generated with the linearized episomal plasmid. Tfrc was used as a loading control for normalization. Due to cross-reactivity of the primers with the endogenous Alb gene, the detection limit of the assay is 2, for 2 Alb copies in the genome.

Fusion mRNA analysis

[000117] Mouse liver RNA was isolated using the RNeasy kit from Qiagen. The copy number of fusion mRNAs was determined by ccPCR with the Fwd/Rγ primer set (FIG. 4). The copy number of Alb was measured by ccPCR with the Fwd/R _E primer set and used for normalization.

Albumin-2A ELISA

[000118] Plasma albumin-2A was measured by chemiluminescence ELISA using a proprietary rabbit polyclonal anti-2A antibody for capture and an HRP-labeled sheep polyclonal anti-albumin antibody (BioRad AHP102P) for detection (FIG. 5A). Recombinant mouse albumin-2A expressed in mammalian cells and affinity purified was used to construct a standard curve in 10% control mouse plasma to account for matrix effects (FIG. 5B). Casein at 1% (Thermo 37528) was used for blocking and at 0.1% for dilution of samples in PBST.

Albumin ELISA

[000119] Total mouse albumin in plasma was measured by chemiluminescence ELISA using polyclonal goat anti-mouse albumin antibody (abcam ab19194) for capture and polyclonal sheep HRP-labeled anti-albumin antibody (BioRad AHP102P) for detection. Mouse albumin standard was purchased from Sigma (SLBX6058). Casein at 1% (Thermo 37528) was used for blocking and at 0.1% for dilution of samples in PBST.

Highly sensitive albumin-2A ELISA

[000120] The original albumin-2A ELISA protocol was optimized to improve assay sensitivity and minimize matrix interference. A proprietary recombinant rabbit monoclonal anti-2A antibody was developed and used for capture and a polyclonal goat anti-albumin antibody labeled with HRP (abcam ab19195) was used for detection (FIG. 5A). Recombinant mouse albumin-2A expressed in mammalian cells and affinity purified to a purity of >95% was used to construct a standard curve in ≤1% control mouse plasma or serum (FIG. 14). Blocking was performed with a buffer consisting of 1% non-fat dry milk and samples were diluted in 1% BSA in PBST. The lower limit of detection using this optimized protocol is <1 ng/mL.

[000121]

Human Factor IX ELISA

[000122] Human factor IX in plasma was measured by chemiluminescence ELISA using monoclonal mouse anti-human factor IX antibody (Sigma F2645) for capture and goat polyclonal HRP-labeled anti-human factor IX antibody (Affinity Biologicals GAFIX-APHRP) for detection. Human factor IX standard was purchased from abcam (ab62544) and a standard curve was generated in 6% control mouse plasma to account for matrix effects. BSA at 3% was used for blocking and at 1% for sample dilution in PBST.

IFA syn A1AT

[000123] Cyno A1AT in plasma was measured by chemiluminescence ELISA using goat polyclonal anti-A1AT antibody for capture (MP Biomedical 55030) and sheep polyclonal HRP-labeled anti-A1AT antibody (abcam ab8768) for detection. Recombinant cynoa1AT expressed in mammalian cells was used to construct a standard curve in 10% control mouse plasma to account for matrix effects. BSA at 3% was used for blocking and at 1% for dilution of samples in PBST.

Western blot MUT

[000124] Frozen liver tissues (~60 mg) were homogenized in lysis buffer (0.5% Igepal-630, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, supplemented with Roche mini-tablet protease inhibitor cocktail) using MP Biomedicals Lysing Matrix D (#116913050) with two cycles of bead beating (20 sec at 3500 rpm per cycle). Liver lysates were cleared by centrifugation and total protein was quantified by BCA assay. Lysates (6 μg/lane) were resolved on 4-12% NuPAGE BisTris midi-gel (Life Technologies) using MES buffer before transfer to nitrocellulose membranes on a Trans-Turbo Blot system (Bio-Rad). After blocking in LI-COR Odyssey Buffer, membranes were incubated with (a) rabbit monoclonal anti-MUT antibody (abcam ab134956) and mouse monoclonal anti-β-actin antibody (abcam ab14128) or (b) rabbit polyclonal anti-2A antibody (proprietary) and mouse polyclonal anti-albumin (abcam ab19194). After incubation with secondary antibodies (anti-rabbit IRDye800CW and anti-mouse IRDye680CT), blots were scanned in the LI-COR Odyssey system and images were analyzed using ImageStudio software.

Transgenes and vectors

[000125] Human factor IX transgene: Codon-optimized human factor IX cDNA with the coding sequence P2A at the 5' end was flanked by homologous arms of 1.3 bp upstream and 1.4 kb downstream to the Alb stop codon (SEQ ID NO: 3).

[000126] Human methylmalonide-CoA mutase transgene: A codon-optimized human MUT cDNA with the P2A coding sequence at the 5' end was flanked by homologous arms of 1 kb upstream and 1 kb downstream to the Alb stop codon (SEQ ID NO: 4).

[000127] Mouse methylmalonyl-CoA mutase transgene: The mouse MUT cDNA with the P2A coding sequence at the 5' end was flanked by homologous arms of 1 kb upstream and 1 kb downstream to the Alb stop codon (SEQ ID NO: 5).

[000128] Cynomolgus monkey alpha-1 antitrypsin transgene (cyno-A1AT): Codon-optimized cynomolgus monkey SERPINA1 cDNA with the P2A coding sequence at the 5' end was flanked by homologous arms of 1 kb (SEQ ID NO: 6) or 750 bp (SEQ ID NO: 7) upstream and downstream of the Alb stop codon.

[000129] Vector preparation: All plasmids for rAAV production were prepared using the EndoFree Plasmid Gigaprep Kit from Qiagen.

[000130] DJ vectors were generated at laboratory scale using triple transfection in adherent HEK-293 cells with CsCl gradient purification for hF9 and MUT and using triple transfection in suspension HEK-293F cells with affinity purification followed by iodixanol gradient for cyno-A1AT. Physical titers were quantified by qPCR

In vivo studies

[000131] All animal procedures were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines for the care and use of animals in research.

[000132] C57BL/6 and FvB/NJ mice were purchased from The Jackson Laboratory to serve as breeding pairs to generate offspring for neonatal (p2) and juvenile (p21) injections. 2-day-old mice were injected intravenously by facial vein with ~10 µl vector for a final dose of 1e13 or 1e14 gv/kg, or PBS for the vehicle group. 21-day-old mice were injected intravenously by tail vein with ~100 µl vector for a final dose of 1e13 or 1e14 gv/kg, or PBS for the vehicle group. Adult mice (>6 weeks of age) were injected intravenously by tail vein with ~200 µl vector for a final dose of 1e13 or 1e14 gv/kg, or PBS for the vehicle group. Animal weights were monitored weekly. Intravital blood collection was performed by buccal bleeding and terminal bleeding by cardiac puncture, except for p7 animals, which were decapitated. At selection for analysis, mice were euthanized by CO ₂ inhalation, the livers were collected, dissected and immediately frozen.

[000133] The MUT (Mut ^−/− ;Tg ^INS-MCK-Mut ) mouse model was obtained from Dr. Charles Venditti (National Human Genome Research Institute, National Institutes of Health). Neonatal mice (p1-2) were injected intravenously into the facial vein with ~10 µl of the hMUT-DJ vector at a final dose of 1e13, 3e13, or 1e14 gv/kg, or PBS as vehicle.

Example 2: Detection of episomal DNA

[000134] This example demonstrates the use of episomal DNA detection and analysis in accordance with the methods disclosed herein.

[000135] Neonatal mice were injected intravenously with a viral vector comprising the P2A coding sequence and a human factor IX transgene designed to integrate into the endogenous albumin target site (see the human factor IX transgene described in Example 1).

[000136] The number of episomal copies decreased exponentially over time after injection (FIG. 6A). The decrease in episomal copy number was observed in parallel with liver growth (from 0.15 g at p7 to 1 g at 8 weeks of age) (FIG. 6B). Despite the decrease in episomal copies per cell over time, transgene expression remained high, as expected after stable integration of the transgene into the genome within the first week, as achieved using GeneRide™ (see, e.g., FIG. 7A and FIG. 8A).

[000137] These data demonstrate that the amount of episomal DNA per cell decreases over time as the liver grows. These data further demonstrate that the growth and development of animals is not affected by high titer dosing of AAV, in this case 1e14 gv/kg (FIGS. 6B-6C). These data also demonstrate that the number of episomal copies of a viral vector delivered in vivo can be a surrogate for dosage and tissue growth. Specifically, the number of episomal copies is proportional to the dose the animal received. For example, if an animal is dosed incorrectly (as can occur when administered intravenously to neonatal mice), the number of episomal copies for that animal will be much lower than for an animal that received a full dose.

Example 3: Detection of ALB-2A in plasma and correlation with changes in endogenous albumin expression

[000138] This example demonstrates the detection and monitoring of albumin labeled with 2A peptide following in vivo delivery of a nucleic acid element encoding 2A peptide. This example also demonstrates that changes in plasma ALB-2A levels can be associated with changes in endogenous albumin expression.

[000139] Neonatal mice were injected intravenously with the viral vector described in Example 2. Genomic integration could be detected as early as 1 week after injection and had already reached a plateau by 2 weeks (FIG. 7A). Blood ALB-2A levels increased during the first 3-4 weeks and then stabilized (FIG. 7B). The observed increase in plasma ALB-2A was associated with an exponential increase in endogenous albumin after birth (FIG. 7C and FIG. 7D).

[000140] These data demonstrate that the methods used in accordance with the present disclosure provide ready-made detection and/or analysis of ALB-2A in plasma, and that detection and/or analysis of ALB-2A in plasma can be achieved relatively early (e.g., within 1 week) after in vivo delivery of a nucleic acid element encoding peptide 2A. These data also demonstrate that plasma ALB-2A levels can be monitored at multiple time intervals after initial delivery of the nucleic acid element encoding peptide 2A, and that plasma ALB-2A levels correlate with changes in the levels of the endogenous polypeptide that is encoded at the target site for peptide 2A integration (e.g., the albumin target site).

Example 4: Early detection and analysis of biomarkers and payloads

[000141] This example demonstrates that the methods used in accordance with the present disclosure allow for early in vivo detection and analysis of a payload following in vivo delivery of a nucleic acid encoding a payload and a nucleic acid encoding a biomarker. This example also demonstrates that the kinetics of payload expression are similar to the kinetics of biomarker expression.

[000142] Neonatal mice were injected intravenously with the viral vector described in Example 2. Analysis of plasma levels of human factor IX, which is expressed from the integrated transgene, revealed similar early kinetics to ALB-2A (FIGS. 8A-8B, respectively). Similar to the kinetics of ALB-2A in plasma, the level of factor IX increased as endogenous albumin increased after birth.

[000143] These data demonstrate that expression of the payload (e.g., factor IX) can be detected and/or analyzed relatively early (e.g., within 1 week) after delivery via a viral vector. These data also demonstrate similar kinetics of payload expression and plasma levels of a biomarker delivered with the payload (e.g., peptide 2A) over multiple time intervals after initial payload delivery.

Example 5: Animal age at dosing does not affect integration efficiency, plasma ALB-2A and transgene

[000144] This example demonstrates that the methods used in accordance with the present disclosure can be used for gene therapy treatments administered at various ages to subjects receiving such treatment, including at very early (e.g., infancy) and developmental stages and into adulthood. This example also demonstrates that the methods described herein can be used to analyze gene therapy that is delivered to tissues of short or tall stature.

[000145] Neonatal (p2) or juvenile (p21) mice were injected intravenously with the viral vector described in Example 2 and collected for analysis at 8 weeks post-injection. Regardless of age at dosing, ALB-2A and human factor IX were readily detected in plasma (FIGS. 9A and 9B, respectively), as was biomarker 2A RNA integration (FIG. 9C). The efficiency of integration into genomic DNA was not significantly affected by the age at which the animals were dosed (FIG. 9D). Episomal copy number was still high at 8 weeks post-injection in animals dosed at the p21 level (FIG. 9E), consistent with lower liver growth in juvenile animals.

[000146] These data demonstrate that expression of a biomarker (e.g., 2A) and a payload (e.g., factor IX) can be detected and/or analyzed following delivery of said biomarker and payload to a subject at different stages of development, including at different ages of the subject and/or at different stages of tissue growth in tissue-directed delivery. In the present example, use of the DJ vector is directed to delivery of the 2A nucleic acid and payload to the liver.

Example 6: Biomarker levels as a proxy for payload levels when administering a drug at different ages

[000147] This example demonstrates that the methods used in accordance with the present disclosure can detect and analyze a biomarker as a surrogate for the level of a payload. This example also demonstrates that using a biomarker as a surrogate can be practiced for delivering a payload and a biomarker in different age groups of subjects.

[000148] Neonatal (p2), juvenile (p21), or adult (p42 p63) mice were injected intravenously with the viral vector described in Example 2 and harvested for analysis at 4 weeks post-injection. Readiness for detection of ALB-2A (FIG. 10A) and human factor IX (FIG. 10B) in plasma was observed in each age group compared to vehicle treatment. Relative levels of ALB-2A were indicative of human factor IX levels among age groups tested (FIG. 10C).

[000149] These data demonstrate that detection of a biomarker (e.g., ALB-2A) is indicative of payload (e.g., factor IX) expression. These data further illustrate that detection and analysis of a biomarker may be useful as a surrogate for the level of payload delivered to a subject. Moreover, such measurement of biomarker levels as a surrogate for the level of payload may be useful for analyzing payload delivered to subjects in different age groups.

Example 7: Biomarker levels as a proxy for payload levels observed with vector design changes

[000150] Neonatal mice were injected intravenously with a viral vector comprising the P2A coding sequence, cynomolgus SERPINA1 cDNA, and 1 kb to 750 bp homology arms designed to integrate into the endogenous albumin target site (see the cynomolgus alpha-1 antitrypsin (cyno-A1AT) transgene described in Example 1). Animals were collected for analysis at 6 weeks post-injection. Plasma levels of ALB-2A (FIG. 11A) were detected following delivery of viral vectors containing the 750 bp and 1 kb homology arms. Plasma levels of cynomolgus A1AT, which is expressed from the integrated transgene, were also readily detectable in both sets of homology arms tested (FIG. 11B). Relative ALB-2A levels were indicative of cynomolgus macaque A1AT levels (FIG. 11C).

Example 8: Plasma biomarker levels as an indirect indicator of intracellular payload integration and expression

In the present example, a mouse model of MMA, termed Mut ^-/- ;Tg ^INS-MCK-Mut mice (referred to herein as MCK-Mut), was used. In this mouse model, a functional copy of the mouse Mut gene is under the control of the creatine kinase promoter, allowing Mut to be expressed in muscle cells. Neonatal MCK-Mut (p2) mice were injected intravenously with various doses of a viral vector containing the P2A coding sequence and a codon-optimized human MUT cDNA (see the human methylmalonyl-CoA mutase transgene described in Example 1). Animals were collected for analysis for 3 months. ALB-2A in plasma and genomic integration in the liver were readily detected (FIG. 12A). The relative levels of ALB-2A in the liver and in plasma were indicative of the level of MUT protein in the liver (FIG. 12B).

[000151] These data demonstrate that the methods used in accordance with the present disclosure allow for the expression of an intracellular payload (e.g., MUT), the expression of which can be assessed by detecting and analyzing the expression of a detectable fragment fused to a polypeptide encoded by a target site gene (e.g., ALB-2A). These data also demonstrate that changes in the expression of the intracellular payload (e.g., MUT) can be reflected in similar changes in the plasma levels of the detectable fragment (e.g., peptide 2A), such that detection of plasma levels of the detectable fragment can act as a surrogate for detecting the intracellular payload. Furthermore, these data demonstrate the ability to monitor intracellular payload levels in real time without the need for detection of the payload itself (e.g., via liver biopsy).

Example 9: Biomarker levels as a proxy for changing payload levels over time

[000152] Neonatal MUT-MCK (p2) mice were injected intravenously with 1e14 gv/kg DJ-hMUT and mice were collected for analysis at 7 months. GeneRide-edited hepatocytes express functional MUT, giving them a selective growth advantage over endogenous Mut ^-/- hepatocytes. As a result, faster expansion of the gene-edited population is expected and observed. This expansion can be detected by increased levels of the transgene as well as ALB-2A (FIG. 13A-FIG. 13B).

[000153] These data indicate that payload levels can increase over time following delivery of a single dose to tissue that lacks expression of the wild-type protein corresponding to the polypeptide encoded by the payload. These data also demonstrate that an increase in biomarker levels is accompanied by an increase in payload levels. One skilled in the art will be able to use the methods disclosed herein to detect and analyze a biomarker in plasma, such as ALB-2A, as a surrogate for intracellular payload expression (e.g., MUT).

Example 10: Long-term detection and analysis of biomarker and payload

[000154] This example demonstrates that the methods used in accordance with the present disclosure allow for sustained in vivo detection and analysis of a payload following in vivo delivery of a nucleic acid encoding the payload and a nucleic acid encoding a biomarker. This example also demonstrates validation of the assay in that the kinetics of expression of the payload are similar to the kinetics of expression of the biomarker.

[000155] Adult mice were injected intravenously with the viral vector described in Example 2. Analysis of plasma levels of human factor IX, which is expressed from the integrated transgene, revealed kinetics similar to ALB-2A from week 1 to week 16 (FIGS. 15A-15B), increasing rapidly during the first weeks after injection until reaching a steady state.

[000156] These data demonstrate that payload (e.g., factor IX) expression can be assessed over an extended period of time (e.g., up to 16 weeks) following delivery via a viral vector. Importantly, these data confirm similar kinetics of payload expression and plasma levels of a biomarker delivered with the payload (e.g., peptide 2A) over multiple time intervals following initial payload delivery.

Example 11: Dose-dependent detection and analysis of biomarker, gDNA and payload

[000157] This example demonstrates that the methods used in accordance with the present disclosure allow for dose-dependent detection and analysis of a payload following in vivo delivery of a nucleic acid encoding a biomarker.

[000158] These data demonstrate that the methods used in accordance with the present disclosure provide dose-dependent expression of a payload, here an intracellular payload (e.g., mMUT) or a secreted payload (e.g., cA1AT), the expression of which can be assessed by detecting and analyzing the expression of a detectable fragment fused to a polypeptide encoded by a target site gene (e.g., ALB-2A). These data also demonstrate that dose-dependent changes in the expression of a secreted payload (e.g., cA1AT) can be reflected in similar changes in the plasma level of a detectable fragment (e.g., peptide 2A), such that detection of a plasma level of a detectable fragment can serve as a surrogate for detection of a secreted payload (FIG. 16A-C). Furthermore, these data demonstrate that genomic integration levels for the intracellular payload can also correlate with similar changes in plasma levels of the detectable fragment (FIG. 17A-C).

Equivalents and scope of application

Those skilled in the art will recognize, or be able to ascertain with no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not limited by the description above, but is in accordance with the claims below:

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SEQUENCE LIST

<110> LogicBio Therapeutics, Inc.

<120> MONITORING GENE THERAPY

<130> 2012538-0082

<150> 62/833875

<151> 04/15/2019

<160> 7

<170> Patent version 3.5

<210> 1

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 1

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 2

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 2

gccaccaact tcagcctgct gaaacaggcc ggcgacgtgg aagagaaccc tggcccc 57

<210> 3

<211> 4172

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

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aggttcgaac cctgctgaag ggagaggttc caatactaca aaatgtagcg ggatattgtc 60

atcacctttg gggacatgtc atcatggtcc cgacacagag ttacaaaact catcccctac 120

acagcactat gtctctggta ctgtttgttc tacagatgtc aacaacagag gcccagccat 180

ctcctattgc ttggcttgtc agtctttcta gcctccccat tattaatttc aaatggggca 240

ggtgttagga gggcaaaaat ccacatatta agtgcaaagc ctttcaggag atttcctgaa 300

actagacaaa acccgtgtga ctggcatcga ttattctatt tgatctagct agtcctagca 360

aagtgacaac tgctactccc ctcctacaca gccaagattc ctaagttggc agtggcatgc 420

ttaatcctca aagccaaagt tacttggctc caagatttat agccttaaac tgtggcctca 480

cattccttcc tatcttactt tcctgcactg gggtaaatgt ctccttgctc ttcttgcttt 540

ctgtcctact gcagggctct tgctgagctg gtgaagcaca agcccaaggc tacagcggag 600

caactgaaga ctgtcatgga tgactttgca cagttcctgg atacatgttg caaggctgct 660

gacaaggaca cctgcttctc gactgaggtc agaaacgttt ttgcattttg acgatgttca 720

gtttccatt tctgtgcacg tggtcaggtg tagctctctg gaactcacac actgaataac 780

tccaccaatc tagatgttgt tctctacgta actgtaatag aaactgactt acgtagcttt 840

taatttttat tttctgccac actgctgcct attaaatacc tattatcact atttggtttc 900

aaatttgtga cacagaagag catagttaga aatacttgca aagcctagaa tcatgaactc 960

atttaaacct tgccctgaaa tgtttctttt tgaattgagt tattttacac atgaatggac 1020

agttaccatt atatatctga atcatttcac attccctccc atggcctaac aacagtttat 1080

cttcttattt tgggcacaac agatgtcaga gagcctgctt taggaattct aagtagaact 1140

gtaattaagc aatgcaaggc acgtacgttt actatgtcat tgcctatggc tatgaagtgc 1200

aaatcctaac agtcctgcta atacttttct aacatccatc atttctttgt tttcagggtc 1260

caaaccttgt cactagatgc aaagacgcct tagccggaag cggcgccacc aatttcagcc 1320

tgctgaaaca ggccggcgac gtggaagaga accctggccc tgctagccag cgcgtgaaca 1380

tgattatggc cgagagccct ggcctgatca ccatctgcct gctgggctac ctgctgagcg 1440

ccgagtgtac cgtgttcctg gaccacgaga acgccaacaa gatcctgaac agacccaaga 1500

gatacaacag cggcaagctg gaagagttcg tgcagggcaa cctggaacgc gagtgcatgg 1560

aagagaagtg cagcttcgaa gaggccagag aggtgttcga gaacaccgag agaaccaccg 1620

agttctggaa gcagtacgtg gacggcgacc agtgcgagag caacccttgt ctgaacggcg 1680

gcagctgcaa ggacgacatc aacagctacg agtgctggtg ccccttcggc ttcgagggca 1740

agaactgcga gctggacgtg acctgcaaca tcaagaacgg cagatgcgag cagttctgca 1800

agaacagcgc cgacaacaag gtcgtgtgct cctgcaccga gggctacaga ctggccgaga 1860

accagaagtc ctgcgagccc gctgtgcctt tcccatgcgg aagagtgtcc gtgtcccaga 1920

ccagcaagct gaccagagcc gagacagtgt tccccgacgt ggactacgtg aacagcaccg 1980

aggccgagac aatcctggac aacatcaccc agagcaccca gtccttcaac gacttcacca 2040

gagtcgtggg cggcgaggat gctaagcctg gccagttccc gtggcaggtg gtgctgaacg 2100

gaaaggtgga cgccttctgc ggcggctcca tcgtgaacga gaagtggatc gtgacagccg 2160

cccactgcgt ggaaaccggc gtgaagatca cagtggtggc cggcgagcac aacatcgagg 2220

aaaccgagca cacagagcag aaaagaaacg tgatcaggat catcccccac cacaactaca 2280

acgccgccat caacaagtac aaccacgata tcgccctgct ggaactggac gagcccctgg 2340

tgctgaatag ctacgtgacc cccatctgta tcgccgacaa agagtacacc aacatctttc 2400

tgaagttcgg cagcggctac gtgtccggct ggggcagagt gtttcacaag ggcagatccg 2460

ctctggtgct gcagtacctg agagtgcctc tggtggacag agccacctgt ctgagaagca 2520

ccaagttcac catctacaac aacatgttct gcgctggctt ccacgagggc ggcagagact 2580

cttgtcaggg cgattctggc ggccctcacg tgacagaggt ggaaggcacc agctttctga 2640

ccggcatcat cagctggggc gaggaatgcg ccatgaaggg gaagtacggc atctacacca 2700

aggtgtccag atacgtgaac tggatcaaag aaaagaccaa gctgacataa gctagcttag 2760

cctaaacaca tcacaaccac aaccttctca ggtaactata cttgggactt aaaaaacata 2820

atcataatca tttttcctaa aacgatcaag actgataacc atttgacaag agccatacag 2880

acaagcacca gctggcactc ttaggtcttc acgtatggtc atcagtttgg gttccattg 2940

tagataagaa actgaacata taaaggtcta ggttaatgca atttacacaa aaggagacca 3000

aaccagggag agaaggaacc aaaattaaaa attcaaacca gagcaaagga gttagccctg 3060

gttttgctct gacttacatg aaccactatg tggagtcctc catgttagcc tagtcaagct 3120

tatcctctgg atgaagttga aaccatatga aggaatattt ggggggtggg tcaaaacagt 3180

tgtgtatcaa tgattccatg tggtttgacc caatcattct gtgaatccat ttcaacagaa 3240

gatacaacgg gttctgtttc aataagtg atccacttcc aaatttctga tgtgccccat 3300

gctaagcttt aacagaattt atcttcttat gacaaagcag cctcctttga aaatatagcc 3360

aactgcacac agctatgttg atcaattttg tttataatct tgcagaagag aattttttaa 3420

aatagggcaa taatggaagg ctttggcaaa aaaattgttt ctccatatga aaacaaaaaa 3480

cttatttttt tattcaagca aagaacctat agacataagg ctatttcaaa attatttcag 3540

ttttagaaag aattgaaagt tttgtagcat tctgagaaga cagctttcat ttgtaatcat 3600

aggtaatatg taggtcctca gaaatggtga gacccctgac tttgacactt ggggactctg 3660

agggaccagt gatgaagagg gcacaactta tatcacacat gcacgagttg gggtgagagg 3720

gtgtcacaac atctatcagt gtgtcatctg cccaccaagt aacagatgtc agctaagact 3780

aggtcatgtg taggctgtct acaccagtga aaatcgcaaa aagaatctaa gaaattccac 3840

atttctagaa aataggtttg gaaaccgtat tccattttac aaaggacact tacatttctc 3900

tttttgtttt ccaggctacc ctgagaaaaa aagacatgaa gactcaggac tcatcttttc 3960

tgttggtgta aaatcaacac cctaaggaac acaaatttct ttaaacattt gacttcttgt 4020

ctctgtgctg caattaataa aaaatggaaa gaatctactc tgtggttcag aactctatct 4080

tccaaaggcg cgcttcaccc tagcagcctc tttggctcag aggaatccct gcctttcctc 4140

ccttcatctc agcagagaat gtagttccac at 4172

<210> 4

<211> 4312

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 4

actccatgaa agtggatttt attatcctca tcatgcagat gagaatattg agacttatag 60

cggtatgcct gagccccaaa gtactcagag ttgcctggct ccaagattta taatcttaaa 120

tgatgggact accatcctta ctctctccat ttttctatac gtgagtaatg ttttttctgt 180

tttttttttt tctttttcca ttcaaactca gtgcacttgt tgagcttgtg aaacacaagc 240

ccaaggcaac aaaagagcaa ctgaaagctg ttatggatga tttcgcagct tttgtagaga 300

agtgctgcaa ggctgacgat aaggagacct gctttgccga ggaggtacta cagttctctt 360

cattttaata tgtccagtat tcatttttgc atgtttggtt aggctaggc ttagggattt 420

atatatcaaa ggaggctttg tacatgtggg acagggatct tattttacaa acaattgtct 480

tacaaaatga ataaaacagc actttgtttt tatctcctgc tctattgtgc catactgtta 540

aatgtttata atgcctgttc tgtttccaaa tttgtgatgc ttatgaatat taataggaat 600

atttgtaagg cctgaaatat tttgatcatg aaatcaaaac attaatttat ttaaacattt 660

acttgaaatg tggtggtttg tgatttagtt gattttatag gctagtggga gaatttacat 720

tcaaatgtct aaatcactta aaattgccct ttatggcctg acagtaactt ttttttattc 780

atttggggac aactatgtcc gtgagcttcc gtccagagat tatagtagta aattgtaatt 840

aaaggatatg atgcacgtga aatcactttg caatcatcaa tagcttcata aatgttaatt 900

ttgtatccta atagtaatgc taatattttc ctaacatctg tcatgtcttt gtgttcaggg 960

taaaaaactt gttgctgcaa gtcaagctgc cttaggctta ggcagcggcg ccaccaactt 1020

cagcctgctg aaacaggccg gcgacgtgga agagaaccct ggccccctga gagccaaaaa 1080

ccagctgttc ctgctgagcc cccactatct gagacaggtc aaagaaagtt ccgggagtag 1140

actgatccag cagagactgc tgcaccagca gcagccactg catcctgagt gggccgctct 1200

ggccaagaaa cagctgaagg gcaaaaaccc agaagacctg atctggcaca ctccagaggg 1260

gatttcaatc aagcccctgt acagcaaaag ggacactatg gatctgccag aggaactgcc 1320

aggagtgaag cctttcaccc gcggacctta cccaactatg tatacctttc gaccctggac 1380

aattcggcag tacgccggct tcagtactgt ggaggaatca aacaagtttt ataaggacaa 1440

catcaaggct ggacagcagg gcctgagtgt ggcattcgat ctggccacac atcgcggcta 1500

tgactcagat aatcccagag tcagggggga cgtgggaatg gcaggagtcg ctatcgacac 1560

agtggaagat actaagattc tgttcgatgg aatccctctg gagaaaatgt ctgtgagtat 1620

gacaatgaac ggcgctgtca ttcccgtgct ggcaaacttc atcgtcactg gcgaggaaca 1680

gggggtgcct aaggaaaaac tgaccggcac aattcagaac gacatcctga aggagttcat 1740

ggtgcggaat acttacattt ttccccctga accatccatg aaaatcattg ccgatatctt 1800

cgagtacacc gctaagcaca tgcccaagtt caactcaatt agcatctccg ggtatcatat 1860

gcaggaagca ggagccgacg ctattctgga gctggcttac accctggcag atggcctgga 1920

atattctcga accggactgc aggcaggcct gacaatcgac gagttcgctc ctagactgag 1980

tttcttttgg ggaattggca tgaactttta catggagatc gccaagatga gggctggccg 2040

gagactgtgg gcacacctga tcgagaagat gttccagcct aagaactcta agagtctgct 2100

gctgcgggcc cattgccaga catccggctg gtctctgact gaacaggacc catataacaa 2160

tattgtcaga accgcaatcg aggcaatggc agccgtgttc ggaggaaccc agagcctgca 2220

cacaaactcc tttgatgagg ccctggggct gcctaccgtg aagtctgcta ggattgcacg 2280

caatacacag atcattatcc aggaggaatc cggaatccca aaggtggccg atccctgggg 2340

aggctcttac atgatggagt gcctgacaaa cgacgtgtat gatgctgcac tgaagctgat 2400

taatgaaatc gaggaaatgg ggggaatggc aaaggccgtg gctgagggca ttccaaaact 2460

gaggatcgag gaatgtgcag ctaggcgcca ggcacgaatt gactcaggaa gcgaagtgat 2520

cgtcggggtg aataagtacc agctggagaa agaagacgca gtcgaagtgc tggccatcga 2580

taacacaagc gtgcgcaatc gacagattga gaagctgaag aaaatcaaaa gctcccgcga 2640

tcaggcactg gccgaacgat gcctggcagc cctgactgag tgtgctgcaa gcggggacgg 2700

aaacattctg gctctggcag tcgatgcctc ccgggctaga tgcactgtgg gggaaatcac 2760

cgacgccctg aagaaagtct tcggagagca caaggccaat gatcggatgg tgagcggcgc 2820

ttatagacag gagttcgggg aatctaaaga gattaccagt gccatcaaga gggtgcacaa 2880

gttcatggag agagaagggc gacggcccag gctgctggtg gcaaagatgg gacaggacgg 2940

acatgatcgc ggagcaaaag tcattgccac cgggttcgct gacctgggat ttgacgtgga 3000

tatcggccct ctgttccaga caccacgaga ggtcgcacag caggcagtcg acgctgatgt 3060

gcacgcagtc ggagtgtcca ctctggcagc tggccataag accctggtgc ctgaactgat 3120

caaagagctg aactctctgg gcagaccaga catcctggtc atgtgcggcg gcgtgatccc 3180

accccaggat tacgaattcc tgtttgaggt cggggtgagc aacgtgttcg gaccaggaac 3240

caggatccct aaggccgcag tgcaggtcct ggatgatatt gaaaagtgtc tggaaaagaa 3300

acagcagtca gtgtaacatc acatttaaaa gcatctcagg taactatatt ttgaattttt 3360

taaaaaagta actataatag ttattattaa aatagcaaag attgaccatt tccaagagcc 3420

atatagacca gcaccgacca ctattctaaa ctatttatgt atgtaaatat tagcttttaa 3480

aattctcaaa atagttgctg agttgggaac cactattatt tctattttgt agatgagaaa 3540

atgaagataa acatcaaagc atagattaag taattttcca aagggtcaaa attcaaaatt 3600

gaaaccaaag tttcagtgtt gcccattgtc ctgttctgac ttatatgatg cggtacacag 3660

agccatccaa gtaagtgatg gctcagcagt ggaatactct gggaattagg ctgaaccaca 3720

tgaaagagtg ctttataggg caaaaacagt tgaatatcag tgatttcaca tggttcaacc 3780

taatagttca actcatcctt tccattggag aatatgatgg atctaccttc tgtgaacttt 3840

atagtgaaga atctgctatt acatttccaa tttgtcaaca tgctgagctt taataggact 3900

tatcttctta tgacaacatt tattggtgtg tccccttgcc tagcccaaca gaagaattca 3960

gcagccgtaa gtctaggaca ggcttaaatt gttttcactg gtgtaaattg cagaaagatg 4020

atctaagtaa tttggcattt attttaatag gtttgaaaaa cacatgccat tttacaaata 4080

agacttatat ttgtcctttt gtttttcagc ctaccatgag aataagagaa agaaaatgaa 4140

gatcaaaagc ttattcatct gtttttcttt ttcgttggtg taaagccaac accctgtcta 4200

aaaaacataa atttctttaa tcattttgcc tcttttctct gtgcttcaat taataaaaaa 4260

tggaaagaat ctaatagagt ggtacagcac tgttattttt caaagatgtg tt 4312

<210> 5

<211> 4307

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 5

ttacttggtg ggcagatgac acactgatag atgttgtgac accctctcac cccaactcgt 60

gcatgtgtga tataagttgt gccctcttca tcactggtcc ctcagagtcc ccaagtgtca 120

aagtcagggg tctcaccatt tctgaggacc tacatattac ctatgattac aaatgaaagc 180

tgtcttctca gaatgctaca aaactttcaa ttctttctaa aactgaaata attttgaaat 240

agccttatgt ctataggttc tttgcttgaa taaaaaaata agttttttgt tttcatatgg 300

agaaacaatt tttttgccaa agccttccat tattgcccta ttttaaaaaa ttctcttctg 360

caagattata aacaaaattg atcaacatag ctgtgtgcag ttggctatat tttcaaagga 420

ggctgctttg tcataagaag ataaattctg ttaaagctta gcatggggca catcagaaat 480

ttggaagtgg atcacttatt atgaaacaga acccgttgta tcttctgttg aaatggattc 540

acagaatgat tgggtcaaac cacatggaat cattgataca caactgtttt gacccacccc 600

ccaaatattc cttcatatgg tttcaacttc atccagagga taagcttgac taggctaaca 660

tggaggactc cacatagtgg ttcatgtaag tcagagcaaa accagggcta actcctttgc 720

tctggtttga atttttaatt ttggttcctt ctctccctgg tttggtctcc ttttgtgtaa 780

attgcattaa cctagacctt tatatgttca gtttcttatc tacaaatgga acccaaactg 840

atgaccatac gtgaagacct aagagtgcca gctggtgctt gtctgtatgg ctcttgtcaa 900

atggttatca gtcttgatcg ttttaggaaa aatgattatg attatgtttt ttaagtccca 960

agtatagtta cctgagaagg ttgtggttgt gatgtgttta cacagactgc tgcttctctg 1020

ccaaacactt ctcaatatca tcaagcactt ggacagcagc tcttggaatc cgggttccag 1080

gaccaaagac gttggaaaca ccaacttcat acagaaattc ataatcctgt ggtggaatca 1140

cgcccccaca catgacaagg atatctggcc gccccagggc ggtgagttct ttgataagct 1200

caggaacgag ggttttatga ccagcagcaa gtgtgctgac acccacagca tggacatctg 1260

catccacagc ttgctgcgcc acttcacggg gagtctgaaa aagagggcct atgtccacat 1320

caaaaccaag atcagcaaat cctgtagcaa tgaccttggc tcccctgtca tggccatctt 1380

gtcccatttt tgccacaaga agacgaggtc tgcgaccttc acgttccatg aatttattaa 1440

ctctcttgat ggcagatgtg atctctttac tttctccaaa ctcctgccga tatgctccac 1500

tcaccatacg atcattagct ttatgctcac caaatacttt tttcaaggca tccgtgattt 1560

ctccaactgt acatcttgca cgagctgcat ccactgccag agccagaata ttgccatctc 1620

cactggcagc acactgggta agtgcactga gacactgctc agccaaagct tgatccctgc 1680

tggatttaat cttcttgagt ttttcaatct gcttcttacg cactgaagtg ttgtcaatgg 1740

ccaggacctc cacagagtct tctttttcca actgatactt atttactcca acaattacct 1800

cagaaccaga atctattcta gcttgtcttc gggcagcaca ttcttcaatg cgaagtttag 1860

ggattccttc agctacagct ttggccattc cacccatttc ttcaacttca tatatcaact 1920

tcagagcagc ctcataaacg tcatttgtga gcgactccat catgtacgac cctccccaag 1980

gatccgccac tttggggatc ccagattctt cctgaatgat gatctgtgtg ttccgagcaa 2040

tccgggcact tttcacagtg ggcaaaccca gcgcttcatc aaaagagttc gtatgcaaag 2100

actgggttcc tccaaacaca gctgccatgg cttcgattgc agtgcggaca atgttattgt 2160

aaggatcctg ctcagtaagt gaccaccccg atgtctggca gtgtgctctt agaagaagag 2220

atttagagtt tttaggctgg aacatttttt ctattaagtg agcccacagt cttctcccag 2280

ctcgcatctt ggctatttcc atgtagaagt tcattccaat tccccagaag aaagacaacc 2340

ttggtgcaaa ttcatcaatt gtgagtccag cctggagtcc agttctgcag tactccaacc 2400

catctgcgat ggtataggcc agttctaaaa tggcatcagc tcctgcttcc tgcatatggt 2460

acccgctaat cgaaatggaa ttaaattttg gcatgtgctg tgctgtgtat tggaaaatgt 2520

cagcaataat tttcatcgat ggctctgggg gaaaaatata agtatttctg accataaact 2580

cctttaggat gtcattctga attgtgccag tgagcttctc cttcggcaca ccttgctctt 2640

ccccagttac tataaatgtt gccaggactg ggatgacagc tccgttcata gtcatggaaa 2700

cggacatttt ttctaaagga atgccatcaa ataggatttt ggtgtcttct acagtgtcaa 2760

tagcaactcc agccattcca acatctccac gaactctggg gttgtctgaa tcataaccac 2820

gatgtgttgc caagtcaaag gcaacagaca acccctgctg accagcctta atattgtcct 2880

tatagaattt attgctttct tccacagtac taaagcctgc atactgacgg atggtccagg 2940

gcctataggt atacatggta ggatatggtc cccgtgtgaa tggcttcact cctggaagtt 3000

cttcgggtaa gtccagagta tctgccctgg aatataaggg ctttatagag atcccttctg 3060

gggtgtgcca tataaggtcc tctgggtttt tgcctttcag ctgctttttg gccagtacag 3120

cccattctgg gtgaaggggt tgctgctggt gtagaaggcg tttccatctg gaagctgatg 3180

gaatgtttag ctgcttcagg taatggggcg atagcaaaaa aagttgattc ttagctctca 3240

aggggccagg gttctcttcc acgtcgccgg cctgtttcag caggctgaag ttggtggcgc 3300

cgctgccggc taaggcgtct ttgcatctag tgacaaggtt tggaccctga aaacaaagaa 3360

atgatggatg ttagaaaagt attagcagga ctgttaggat ttgcacttca tagccatagg 3420

caatgacata gtaaacgtac gtgccttgca ttgcttaatt acagttctac ttagaattcc 3480

taaagcaggc tctctgacat ctgttgtgcc caaaataaga agataaactg ttgttaggcc 3540

atgggaggga atgtgaaatg attcagatat ataatggtaa ctgtccattc atgtgtaaaa 3600

taactcaatt caaaaagaaa catttcaggg caaggtttaa atgagttcat gattctaggc 3660

tttgcaagta tttctaacta tgctcttctg tgtcacaaat ttgaaaccaa atagtgataa 3720

taggtattta ataggcagca gtgtggcaga aaataaaaat taaaagctac gtaagtcagt 3780

ttctattaca gttacgtaga gaacaacatc tagattggtg gagttattca gtgtgtgagt 3840

tccagagagc tacacctgac cacgtgcaca gaaaatggaa actgaacatc gtcaaaatgc 3900

aaaaacgttt ctgacctcag tcgagaagca ggtgtccttg tcagcagcct tgcaacatgt 3960

atccaggaac tgtgcaaagt catccatgac agtcttcagt tgctccgctg tagccttggg 4020

cttgtgcttc accagctcag caagagccct gcagtaggac agaaagcaag aagagcaagg 4080

agacatttac cccagtgcag gaaagtaaga taggaaggaa tgtgaggcca cagtttaagg 4140

ctataaatct tggagccaag taactttggc tttgaggatt aagcatgcca ctgccaactt 4200

aggaatcttg gctgtgtagg aggggagtag cagttgtcac tttgctagga ctagctagat 4260

caaatagaat aatcgatgcc agtcacacgg gttttgtcta gtttcag 4307

<210> 6

<211> 3344

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 6

ctgaaactag acaaaacccg tgtgactggc atcgattatt ctatttgatc tagctagtcc 60

tagcaaagtg acaactgcta ctcccctcct acacagccaa gattcctaag ttggcagtgg 120

catgcttaat cctcaaagcc aaagttactt ggctccaaga tttatagcct taaactgtgg 180

cctcacattc cttcctatct tactttcctg cactggggta aatgtctcct tgctcttctt 240

gctttctgtc ctactgcagg gctcttgctg agctggtgaa gcacaagccc aaggctacag 300

cggagcaact gaagactgtc atggatgact ttgcacagtt cctggataca tgttgcaagg 360

ctgctgacaa ggacacctgc ttctcgactg aggtcagaaa cgtttttgca ttttgacgat 420

gttcagtttc cattttctgt gcacgtggtc aggtgtagct ctctggaact cacacactga 480

ataactccac caatctagat gttgttctct acgtaactgt aatagaaact gacttacgta 540

gcttttaatt tttattttct gccacactgc tgcctattaa atacctatta tcactatttg 600

gtttcaaatt tgtgacacag aagagcatag ttagaaatac ttgcaaagcc tagaatcatg 660

aactcattta aaccttgccc tgaaatgttt ctttttgaat tgagttattt tacacatgaa 720

tggacagtta ccattatata tctgaatcat ttcacattcc ctcccatggc ctaacaacag 780

tttatcttct tattttgggc acaacagatg tcagagagcc tgctttagga attctaagta 840

gaactgtaat taagcaatgc aaggcacgta cgtttactat gtcattgcct atggctatga 900

agtgcaaatc ctaacagtcc tgctaatact tttctaacat ccatcatttc tttgttttca 960

gggtccaaac cttgtcacta gatgcaaaga cgccttagcc ggcagcggcg ccaccaactt 1020

cagcctgctg aaacaggccg gcgacgtgga agagaaccct ggcccctctt ctgtctcatg 1080

gggcgtcctc ctgctggctg gcctgtgctg cctgctcccc ggctctctgg ctgaggatcc 1140

ccaggggagat gctgcccaaa aaacggatac atccctccat gatcaagacc acccaaccct 1200

caacaagatc acccccagcc tggctgagtt cggcttcagc ctataccgcc agctggcaca 1260

ccagtccaac agcaccaata tcttcttctc cccagtgagc atcgctacag cctttgcaat 1320

gctctccctg gggaccaagg ctgacactca cagtgaaatc ctggagggcc tgaatttcaa 1380

cgtcacggag attccggagg ctcaggtcca tgaaggcttc caggaactcc tccataccct 1440

caacaagcca gacagccagc tccagctgac caccggcaac ggcctgttcc tcaacaagtc 1500

actcaaagta gtggataagt ttttggagga tgtcaaaaaa ctgtaccact cagaagcctt 1560

ctctgtcaac tttgaggaca ccgaagaggc caagaaacag atcaacaatt acgtggagaa 1620

ggaaactcaa gggaaaattg tggatttggt caaggagctt gacagagaca cagtttttgc 1680

tctggtgaat tacatcttct ttaaaggcaa atgggagaga ccctttgacg ttgaggccac 1740

caaggaagag gacttccacg tggaccaggc gaccaccgtg aaggtgccca tgatgaggcg 1800

tttaggcatg tttaacatct accactgtga gaagctgtcc agctgggtgc tgctgatgaa 1860

atacctgggc aatgccaccg ccatcttctt cctgcctgat gaggggaaac tgcagcacct 1920

ggaaaatgaa ctcacccatg atatcatcac caagttcctg gaaaatgaaa acagcaggtc 1980

tgccaactta catttaccca gactggccat tactggaacc tatgatctga agacagtcct 2040

gggccacctg ggtatcacta aggtcttcag caatggggct gacctctcag ggatcacgga 2100

ggaggcaccc ctgaagctct ccaaggccgt gcataaggct gtgctgacca tcgatgagaa 2160

agggactgaa gctgctgggg ccatgttttt agaggccata cccatgtcta ttccccccga 2220

ggtcaagttc aacaaaccct ttgtcttctt aatgattgaa caaaatacca agtctcccct 2280

cttcatggga aaagtggtga atcccaccca gaaagagcag aagctgatca gcgaggagga 2340

cctgtaaaca catcacaacc acaaccttct caggtaacta tacttgggac ttaaaaaaca 2400

taatcataat catttttcct aaaacgatca agactgataa ccatttgaca agagccatac 2460

agacaagcac cagctggcac tcttaggtct tcacgtatgg tcatcagttt gggttccatt 2520

tgtagataag aaactgaaca tataaaggtc taggttaatg caatttacac aaaaggagac 2580

caaaccaggg agagaaggaa ccaaaattaa aaattcaaac cagagcaaag gagttagccc 2640

tggttttgct ctgacttaca tgaaccacta tgtggagtcc tccatgttag cctagtcaag 2700

cttatcctct ggatgaagtt gaaaccatat gaaggaatat ttggggggtg ggtcaaaaca 2760

gttgtgtatc aatgattcca tgtggtttga cccaatcatt ctgtgaatcc atttcaacag 2820

aagatacaac gggttctgtt tcataataag tgatccactt ccaaatttct gatgtgcccc 2880

atgctaagct ttaacagaat ttatcttctt atgacaaagc agcctccttt gaaaatatag 2940

ccaactgcac acagctatgt tgatcaattt tgtttataat cttgcagaag agaatttttt 3000

aaaatagggc aataatggaa ggctttggca aaaaaattgt ttctccatat gaaaacaaaa 3060

aacttatttt tttattcaag caaagaacct atagacataa ggctatttca aaattatttc 3120

agttttagaa agaattgaaa gttttgtagc attctgagaa gacagctttc atttgtaatc 3180

ataggtaata tgtaggtcct cagaaatggt gagacccctg actttgacac ttggggactc 3240

tgagggacca gtgatgaaga gggcacaact tatatcacac atgcacgagt tggggtgaga 3300

gggtgtcaca acatctatca gtgtgtcatc tgcccaccaa gtaa 3344

<210> 7

<211> 2844

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 7

ctactgcagg gctcttgctg agctggtgaa gcacaagccc aaggctacag cggagcaact 60

gaagactgtc atggatgact ttgcacagtt cctggataca tgttgcaagg ctgctgacaa 120

ggacacctgc ttctcgactg aggtcagaaa cgtttttgca ttttgacgat gttcagtttc 180

cattttctgt gcacgtggtc aggtgtagct ctctggaact cacacactga ataactccac 240

caatctagat gttgttctct acgtaactgt aatagaaact gacttacgta gcttttaatt 300

tttattttct gccacactgc tgcctattaa atacctatta tcactatttg gtttcaaatt 360

tgtgacacag aagagcatag ttagaaatac ttgcaaagcc tagaatcatg aactcattta 420

aaccttgccc tgaaatgttt ctttttgaat tgagttattt tacacatgaa tggacagtta 480

ccattatata tctgaatcat ttcacattcc ctcccatggc ctaacaacag tttatcttct 540

tattttgggc acaacagatg tcagagagcc tgctttagga attctaagta gaactgtaat 600

taagcaatgc aaggcacgta cgtttactat gtcattgcct atggctatga agtgcaaatc 660

ctaacagtcc tgctaatact tttctaacat ccatcatttc tttgttttca gggtccaaac 720

cttgtcacta gatgcaaaga cgccttagcc ggcagcggcg ccaccaactt cagcctgctg 780

aaacaggccg gcgacgtgga agagaaccct ggcccctctt ctgtctcatg gggcgtcctc 840

ctgctggctg gcctgtgctg cctgctcccc ggctctctgg ctgaggatcc ccagggat 900

gctgcccaaa aaacggatac atccctccat gatcaagacc acccaaccct caacaagatc 960

acccccagcc tggctgagtt cggcttcagc ctataccgcc agctggcaca ccagtccaac 1020

agcaccaata tcttcttctc cccagtgagc atcgctacag cctttgcaat gctctccctg 1080

gggaccaagg ctgacactca cagtgaaatc ctggagggcc tgaatttcaa cgtcacggag 1140

attccggagg ctcaggtcca tgaaggcttc caggaactcc tccataccct caacaagcca 1200

gacagccagc tccagctgac caccggcaac ggcctgttcc tcaacaagtc actcaaagta 1260

gtggataagt ttttggagga tgtcaaaaaa ctgtaccact cagaagcctt ctctgtcaac 1320

tttgaggaca ccgaagaggc caagaaacag atcaacaatt acgtggagaa ggaaactcaa 1380

gggaaaattg tggatttggt caaggagctt gacagagaca cagtttttgc tctggtgaat 1440

tacatcttct ttaaaggcaa atgggagaga ccctttgacg ttgaggccac caaggaagag 1500

gacttccacg tggaccaggc gaccaccgtg aaggtgccca tgatgaggcg tttaggcatg 1560

tttaacatct accactgtga gaagctgtcc agctgggtgc tgctgatgaa atacctgggc 1620

aatgccaccg ccatcttctt cctgcctgat gaggggaaac tgcagcacct ggaaaatgaa 1680

ctcacccatg atatcatcac caagttcctg gaaaatgaaa acagcaggtc tgccaactta 1740

catttaccca gactggccat tactggaacc tatgatctga agacagtcct gggccacctg 1800

ggtatcacta aggtcttcag caatggggct gacctctcag ggatcacgga ggaggcaccc 1860

ctgaagctct ccaaggccgt gcataaggct gtgctgacca tcgatgagaa agggactgaa 1920

gctgctgggg ccatgttttt agaggccata cccatgtcta ttccccccga ggtcaagttc 1980

aacaaaccct ttgtcttctt aatgattgaa caaaatacca agtctcccct cttcatggga 2040

aaagtggtga atcccaccca gaaagagcag aagctgatca gcgaggagga cctgtaaaca 2100

catcacaacc acaaccttct caggtaacta tacttgggac ttaaaaaaca taatcataat 2160

catttttcct aaaacgatca agactgataa ccatttgaca agagccatac agacaagcac 2220

cagctggcac tcttaggtct tcacgtatgg tcatcagttt gggttccatt tgtagataag 2280

aaactgaaca tataaaggtc taggttaatg caatttacac aaaaggagac caaaccaggg 2340

agagaaggaa ccaaaattaa aaattcaaac cagagcaaag gagttagccc tggttttgct 2400

ctgacttaca tgaaccacta tgtggagtcc tccatgttag cctagtcaag cttatcctct 2460

ggatgaagtt gaaaccatat gaaggaatat ttggggggtg ggtcaaaaca gttgtgtatc 2520

aatgattcca tgtggtttga cccaatcatt ctgtgaatcc atttcaacag aagatacaac 2580

gggttctgtt tcataataag tgatccactt ccaaatttct gatgtgcccc atgctaagct 2640

ttaacagaat ttatcttctt atgacaaagc agcctccttt gaaaatatag ccaactgcac 2700

acagctatgt tgatcaattt tgtttataat cttgcagaag agaatttttt aaaatagggc 2760

aataatggaa ggctttggca aaaaaattgt ttctccatat gaaaacaaaa aacttatttt 2820

tttattcaag caaagaacct atag 2844

<---

Claims (24)

1. A construct for use in measuring the expression of a therapeutic payload, comprising a 5' to 3' polynucleotide sequence encoding (a) a 5' homologous arm, (b) a 2A coding sequence encoding a 2A peptide, wherein the 2A coding sequence comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, (c) a therapeutic payload, and (d) a 3' homologous arm, wherein said construct integrates into an endogenous albumin target site such that the albumin locus is transcribed to produce albumin-2A and the therapeutic payload as separate proteins; wherein the peptide 2A functions as a biomarker for measuring the expression of said therapeutic payload. 2. A construct for use according to claim 1, wherein the 5'- and 3'-homologous arms of the construct have a length of 1 kb each. 3. A construct for use according to claim 1 or 2, wherein the therapeutic payload of the construct encodes phenylalanine hydroxylase (PAH), glucose-6-phosphatase catalytic subunit (G6PC), propionyl-CoA carboxylase, subunit alpha (PCCA), ATP binding cassette transporter, subfamily B member 11 (ABCB11), ornithine carbamoyltransferase (OTC), UDP-glucuronosyltransferase, family 1, member A1 (UGT1A1), acid alpha-glucosidase (GAA), lysosomal acid glucosylceramidase (GBA), frataxin (FTX), factor IX (F9), or alpha-1-antitrypsin (SERPINA1). 4. A construct for use according to any one of claims 1 to 3, wherein the therapeutic payload of the construct encodes a peptide expressed intracellularly. 5. A construct for use according to any one of claims 1 to 4, wherein the therapeutic payload of the construct encodes a peptide secreted extracellularly. 6. A construct for use according to any one of claims 1 to 5, wherein the therapeutic payload of the construct encodes a peptide having intracellular or extracellular activity that promotes a biological process for treating a medical condition. 7. A construct for use according to any one of claims 1 to 6, wherein the therapeutic payload of the construct encodes a peptide that is normally expressed in liver cells. 8. A construct for use according to any one of claims 1 to 7, wherein the therapeutic payload of the construct encodes a peptide that is ectopically expressed in liver cells. 9. A construct for use according to any one of claims 1 to 8, wherein integration of the construct does not substantially disrupt the expression of albumin encoded at the target site. 10. A structure for use according to any one of claims 1 to 9, wherein the structure is in an AAV vector. 11. A method for monitoring gene therapy, comprising: (i) administering to a subject a gene therapy construct comprising a 5' to 3' polynucleotide sequence encoding (a) a 5' homology arm, (b) a 2A coding sequence encoding a 2A peptide, wherein the 2A coding sequence comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, (c) a therapeutic payload, and (d) a 3' homology arm, wherein said gene therapy construct integrates into an endogenous albumin target site such that the albumin locus is transcribed to produce albumin-2A and the therapeutic payload as separate proteins; and (ii) detecting the expression of the therapeutic payload by measuring albumin-2A in a biological sample from a subject who has received the gene therapy construct, where albumin-2A serves as a biomarker for monitoring gene therapy. 12. The method according to claim 11, wherein the 5’- and 3’-homologous arms have a length of 1 kb each. 13. The method of claim 11 or 12, wherein the therapeutic payload of the gene therapy construct encodes phenylalanine hydroxylase (PAH), glucose-6-phosphatase catalytic subunit (G6PC), propionyl-CoA carboxylase, subunit alpha (PCCA), ATP binding cassette transporter, subfamily B member 11 (ABCB11), ornithine carbamoyltransferase (OTC), UDP-glucuronosyltransferase, family 1, member A1 (UGT1A1), acid alpha-glucosidase (GAA), lysosomal acid glucosylceramidase (GBA), frataxin (FTX), factor IX (F9), or alpha-1-antitrypsin (SERPINA1). 14. The method according to any one of claims 11-13, wherein the therapeutic payload encodes a peptide expressed intracellularly. 15. The method according to any one of claims 11-14, wherein the therapeutic payload encodes a peptide secreted extracellularly. 16. The method according to any one of claims 11-15, wherein the therapeutic payload encodes a peptide having intracellular or extracellular activity that facilitates a biological process for treating a medical condition. 17. The method according to any one of claims 11-16, wherein the therapeutic payload encodes a peptide that is normally expressed in liver cells. 18. The method according to any one of claims 11-17, wherein the therapeutic payload encodes a peptide that is ectopically expressed in liver cells. 19. The method according to any one of claims 11 to 18, wherein integration of the gene therapy construct does not substantially disrupt the expression of albumin encoded at the target site. 20. The method according to any one of claims 11-19, wherein the gene therapy construct is in an AAV vector.