WO2024211447A1 - Tp508 mrnas - Google Patents

Abstract

In one aspect, a platform or construct is configured as a linear nucleic acid DNA or mRNA with an ORF encoding TP508, the nucleic acid producing a TP508 peptide when introduced into a cell.

Description

TP508 MRNAS

RELATED APPLICATIONS

[0001] This application is an International Application claiming priority to U.S. Provisional Patent Application serial number 63/456,601 filed April 3, 2023 which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] None.

REFERENCE TO SEQUENCE LISTING

[0003] A sequence listing required by 37 CFR 1.821-1.825 is being submitted electronically with this application. The sequence listing is incorporated herein by reference.

BACKGROUND

[0004] TP508 is an injectable drug used to mitigate vascular damage, hemorrhage, and inflammation observed in acute respiratory distress syndrome (ARDS) induced by viral infection. TP508 also activates stem cells to regenerate tissues and restore function. Because TP508 can affect cellular processes involved in ARDS progression at multiple points, it has the advantage of potentially showing efficacy where previous candidate drugs have failed.

[0005] Gene delivery for therapy or other purposes is well-known, particularly for the treatment of diseases such as cystic fibrosis and certain cancers. The term refers to the delivery into a cell of a gene or part of a gene to correct some deficiency. In the present specification the term is used also to refer to any introduction of nucleic acid material into target cells and includes the production of peptides in vivo.

[0006] In vivo and in vitro cell delivery systems for expression of proteins or peptides fall into three broad classes, namely those that involve direct injection of naked DNA or RNA, those that make use of viruses or genetically modified viruses and those that make use of non-viral delivery agents. Each has its advantages and disadvantages. Although viruses as delivery agents have the advantages of high efficiency and high cell selectivity, they have the disadvantages of toxicity, production of inflammatory responses and difficulty in dealing with large DNA fragments.

[0007] Non-viral gene delivery systems are based on the compaction of genetic material into nanometric particles by electrostatic interaction between the negatively charged phosphate backbone of DNA or RNA and cationic lipids, peptides or other polymers. The use of non-viral transfection vectors that include lipids, as opposed to viruses, can result in lower toxicity, greater safety, reduced cost, reasonably efficient targeting, and an enhanced packaging ability. Unfortunately, lower transfection efficiencies have been noted particularly with mRNA.

[0008] There is a need for improved and effective delivery for TP508 peptides.

SUMMARY

[0009] Embodiments include but are not limited to nucleic acid platforms or constructs configured for expression of TP508 or TP508 fusion proteins.

[00010] In one aspect, a platform or construct is configured as a linear nucleic acid DNA or RNA with an ORF encoding TP508, the nucleic acid producing a TP508 peptide when introduced into a cell. In certain aspects, the linear nucleic acid platform or construct includes a promoter appropriately positioned 5’ to the open reading frame (ORF). The promoter can be a T7 promoter for example. The linear nucleic acid platform or construct can also include a 5’ UTR that can be positioned between the promoter and 5’ end of the ORF. As a non-limiting example, a UTR can have a sequence of nucleotides CTAGTAGTAGACTCCGCAAGAAGAAGCAAAAAATTAAAGAAGTGAGTTTAAA SEQ ID NO:15. In certain aspects the ORF can encode a TP508 peptide or a TP508 fusion. The encoded peptide can include a N-terminal signal peptide. The nucleic acid platform or construct can include a 3’ UTR. An example of a 3’ UTR is encoded by CAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAA CAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCA GGGTTGGTCAATTTCGTGCCAGCCACACCCTGGTACTGCATGCACGCAATGCTAGCT GCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGC TCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCAAA SEQ ID N0:16. The 3’ terminus can include a poly adenylation segment. A poly adenylation segment can include 20, 40, 50, 60, 70, 80, 90, 100, 110, 120 or more adenine nucleotides.

[00011] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[00012] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[00013] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[00014] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”

[00015] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[00016] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a chemical composition and/or method that “comprises” a list of elements (e.g., components or features or steps) is not necessarily limited to only those elements (or components or features or steps) but may include other elements (or components or features or steps) not expressly listed or inherent to the chemical composition and/or method.

[00017] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[00018] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a chemical composition and/or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel character! stic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[00019] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

[00020] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein. [00021] FIG. 1. Schematic of a representative TP508 mRNA having a 5’ cap, 5’ UTR, TP508 ORF, 3’ UTR, and poly A tail (pA).

DESCRIPTION

[00022] The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be an example of that embodiment, and not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.

[00023] A current interest in the fields of therapeutics and diagnostics is the ability and methods for designing, synthesizing, and delivering a nucleic acid to effect physiologic outcomes beneficial to a cell, a tissue, an organ and ultimately to a subject. The nucleic acid can be a ribonucleic acid (RNA) such as a messenger RNA (mRNA) encoding a peptide or polypeptide of interest. One beneficial outcome is the intracellular translation of the nucleic acid and production of at least one encoded peptide or polypeptide of interest.

[00024] Of particular interest, is the ability to design, synthesize and deliver a nucleic acid, such as a ribonucleic acid (RNA) which encodes a therapeutic protein or peptide. Unlike DNA- based drugs, mRNA transcripts have a relatively high transfection efficiency and low toxicity because they do not need to enter the nucleus to be functional. mRNA presents no risk of accidental infection or opportunistic insertional mutagenesis. In addition, mRNA has broad potential for treating diseases requiring protein expression and higher therapeutic efficacy due to its continuous translation into encoded proteins/peptides to trigger long-lasting expression compared to transient traditional protein/peptide drugs.

[00025] Described herein are compositions (including pharmaceutical compositions) and methods for the design, preparation, manufacture, formulation, and/or use of therapeutic nucleic acids. In particular, described herein are compositions (including pharmaceutical compositions) and methods for the selection, design, preparation, manufacture, formulation, and/or use of nucleic acid (NAs) where at least one component of the NA is a polynucleotide, a DNA polynucleotide, a RNA polynucleotide, and/or a mRNA which encodes a therapeutic peptide. Also provided are systems, processes, devices and kits for the selection, design and/or utilization of the NAs described herein.

I. TP508 Nucleic Acids (NAs)

[00026] Nucleic Acids (NAs) described herein comprise one or more polynucleotides (platform or construct) which encode one or more therapeutic peptides, e.g., TP508 or a TP508 derivative. Polynucleotide constructs include peptide-encoding RNA polynucleotides such as mRNAs. The polynucleotide constructs can include at least one chemical modification. The sequences provided can be the sense strand of a sequence but one of skill would readily identify the complementary anti-sense sequence as well. Also, the nucleotide sequences may be presented as DNA sequences, deoxyribose adenine, guanine, thymine, cytosine (AGTC) and/or RNA sequences ribose adenine, guanine, uracil, cytosine (AGUC); one of skill would readily identify the RNA or DNA counterpart.

[00027] An “effective amount” of the NA composition is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and extent of modified nucleosides) and other components of the NA, and other determinants. In general, an effective amount of the NA composition provides for peptide production in a cell.

[00028] In one embodiment, the polynucleotides of the NAs of the invention may be administrated with other prophylactic or therapeutic compounds.

[00029] In certain aspects, the polynucleotides of the NAs of the invention may be administered intranasally, intramuscularly, intradermally, by instillation (e.g., intratracheal), or by oral or nasal inhalation.

[00030] In certain aspects, the polynucleotides of the NAs of the invention can be transfected ex vivo into cells, which are subsequently transplanted or injected into a subject. In particular aspects the cell are injected into a target tissue or intravenously. II. TP508 NAV Polynucleotides

[00031] According to certain embodiments, the polynucleotides encode at least one polypeptide of interest (a therapeutic peptide), such as TP508 or its derivatives. Peptides of the present invention may be wild type or modified/engineered. They may have any combination of the features described herein.

[00032] The term “nucleic acid,” in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides. These polymers are often referred to as polynucleotides. Nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), which may or may not include ribonucleotide analogs or modifications.

[00033] In some embodiments, the polynucleotide includes from about from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1.000 to 3,000, from 1,000 to 5,000, from 1,500 to 3,000, from 1,500 to 5,000, from 2,000 to 3,000, from 2,000 to 5,000 nucleotides.

[00034] In one embodiment, the polynucleotides of the present invention may encode at least one peptide or polypeptide of interest, e.g., a TP508 or a TP508 derivative.

[00035] In one embodiment, the length of a region encoding at least one polypeptide of interest of the polynucleotides present invention is greater than about 18 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000 nucleotides). As used herein, such a region may be referred to as a “coding region” or “region encoding” or “open reading frame (ORF)”.

[00036] In one embodiment, the polynucleotides of the present invention is or functions as a messenger RNA (mRNA). As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide which encodes at least one peptide or polypeptide of interest and which is capable of being translated to produce the encoded peptide polypeptide of interest in vitro, in vivo, in situ or ex vivo.

[00037] In one embodiment, the polynucleotides of the present invention may be structurally modified or chemically modified. As used herein, a “structural” modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to affect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”. The same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”. Here, the dinucleotide “CC” has been inserted, resulting in a structural modification to the polynucleotide.

[00038] In certain aspects, the polynucleotides have a uniform chemical modification of all or any of the same nucleoside type or a population of modifications, or a measured percent of a chemical modification of all any of the same nucleoside type but with random incorporation, such as where all uridines are replaced by a uridine analog, e.g., pseudouridine. In another embodiment, the polynucleotides may have a uniform chemical modification of two, three, or four of the same nucleoside type throughout the entire polynucleotide (such as all uridines and all cytosines, etc. are modified in the same way).

[00039] When the polynucleotides of the present invention are chemically and/or structurally modified the polynucleotides may be referred to as “modified polynucleotides.”

[00040] Polynucleotide Architecture. Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5^Z UTR, a 3^Z UTR, a 5^Z cap and a poly-A tail. The polynucleotides described herein may function as mRNA and include one or more of these elements.

[00041] In order to further enhance protein production, polynucleotides of the present invention can be designed to be conjugated to other polynucleotides, dyes, intercalating agents (e.g., acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG- 40K), MPEG, [MPEG], polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a lung cell, endothelial cell, or bone cell, hormones and hormone receptors, non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug. In a preferred embodiment, the polynucleotides of the present invention which encode a peptide are conjugated to one or more cell markers. Conjugation may result in increased stability and/or half-life and may be particularly useful in targeting the polynucleotides to specific sites in the cell, tissue or organism.

[00042] As used herein, “polypeptide” or “peptide” means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In one embodiment, the polypeptides of interest are peptides encoded by the polynucleotides as described herein.

[00043] “Substitutional variants” when referring to polypeptides or peptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.

[00044] As used herein the term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.

[00045] “Insertional variants” when referring to polypeptides or peptides are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. “Immediately adjacent” to an amino acid means connected to either the alpha-carboxy or alpha-amino functional group of the amino acid.

[00046] “Deletional variants” when referring to polypeptides or peptides are those with one or more amino acids in the native or starting amino acid sequence removed. Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule.

[00047] “Covalent derivatives” when referring to polypeptides or peptides include modifications of a native or starting protein with an organic proteinaceous or non-proteinaceous derivatizing agent, and/or post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side-chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. The resultant covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays, or for the preparation of antiprotein antibodies for immunoaffinity purification of the recombinant glycoprotein. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.

[00048] As used herein the terms “termini” or “terminus” when referring to polypeptides or peptides refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions. The polypeptide based molecules of the present invention may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins of the invention are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of proteins will have multiple N- and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.

[00049] In some embodiments, the encoded polypeptide variant may have the same or a similar activity as TP508. Generally, variants of TP508 will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and less than or equal to 100% sequence identity to TP508 polynucleotide or peptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSLBLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402.) Other tools are described herein, specifically in the definition of “Identity.” Default parameters in the BLAST algorithm include, for example, an expect threshold of 10, Word size of 28, Match/Mismatch Scores 1, -2, Gap costs Linear. Any filter can be applied as well as a selection for species specific repeats, e.g., Homo sapiens.

[00050] Cell-Penetrating Polypeptides. The polynucleotides disclosed herein may also encode or be conjugated to one or more cell-penetrating polypeptides. As used herein, “cell-penetrating polypeptide” or CPP refers to a polypeptide which may facilitate the cellular uptake of molecules. The cell-penetrating peptide may also include a signal sequence. As used herein, a “signal sequence” refers to a sequence of amino acid residues bound at the amino terminus of a nascent protein during protein translation. The signal sequence may be used to signal the secretion of the peptide or cell-penetrating peptide.

[00051] In one embodiment, the polynucleotides may also encode a fusion protein/peptide. The fusion protein/peptide may be created by operably linking a heterologous protein or peptide to a therapeutic protein/peptide. As used herein, “operably linked” refers to the therapeutic protein and the heterologous protein or peptide being connected in such a way to permit the expression of the complex when introduced into the cell. Preferably, the therapeutic protein may be covalently linked to the heterologous protein or peptide in the formation of the fusion protein. Examples include, but are not limited to FLAG-TP508 or green fluorescent protein (GFP)- TP508.

[00052] Polynucleotides Having Untranslated Regions (UTRs). The polynucleotides of the present invention (e.g., peptide-encoding polynucleotides featured in the NAs of the invention) may comprise one or more regions or parts which act or function as an untranslated region. Where polynucleotides are designed to encode at least one polypeptide of interest, the polynucleotides may comprise one or more of these untranslated regions.

[00053] By definition, untranslated regions (UTRs) of a gene are transcribed but not translated. In mRNA, the 5^Z UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas the 3^Z UTR starts immediately following the stop codon and continues until the transcriptional termination signal. The regulatory features of UTR can be incorporated into the polynucleotides of the present invention to among other things, enhance the stability of the molecule.

[00054] Natural 5^Z UTRs bear features which play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. 5^Z UTR also have been known to form secondary structures which are involved in elongation factor binding. By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of the polynucleotides of the invention.

[00055] Other non-UTR sequences may also be used as regions or subregions within the polynucleotides. For example, introns or portions of introns sequences may be incorporated into regions of the polynucleotides of the invention. Incorporation of intronic sequences may increase protein production as well as polynucleotide levels. [00056] Combinations of features may be included in flanking regions and may be contained within other features. For example, the ORF may be flanked by a 5^Z UTR which may contain a strong Kozak translational initiation signal and/or a 3^Z UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail. 5^Z UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes.

[00057] A UTR from various gene(s) may be incorporated into the regions of the polynucleotide. Furthermore, multiple UTRs of any known gene may be utilized. It is also within the scope of the present invention to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5 ' or 3^Z UTR may be inverted, shortened, lengthened, made with one or more other 5^Z UTRs or 3^Z UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3^Z or 5^Z UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an altered” UTR (whether 3 ' or 5^Z ) comprise a variant UTR.

[00058] In one embodiment, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature of property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.

[00059] 3 ^z UTR and the AU Rich Elements. Natural or wild type 3^Z UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes: Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class.

[00060] Regions Having a 5⁷ Cap. The 5^Z cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5^Z proximal introns removal during mRNA splicing.

[00061] Endogenous mRNA molecules may be 5^Z -end capped generating a 5^Z -ppp-5^z - triphosphate linkage between a terminal guanosine cap residue and the 5^Z -terminal transcribed sense nucleotide of the mRNA molecule. This 5^Z -guanylate cap may then be methylated to generate an N7-methyl -guanylate residue. The ribose sugars of the terminal and/or ante-terminal transcribed nucleotides of the 5^Z end of the mRNA may optionally also be 2^Z -O-methylated. 5 ^z -decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[00062] In some embodiments, polynucleotides may be designed to incorporate a cap moiety. Modifications to the polynucleotides of the present invention may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5^Z -ppp-5^z phosphorodi ester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.) may be used with a -thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5^Z -ppp-5^z cap. Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides. [00063] Additional modifications include, but are not limited to, 2^Z -O-methylation of the ribose sugars of 5^Z -terminal and/or 5^Z -anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2^Z -hydroxyl group of the sugar ring. Multiple distinct 5 ^Z -cap structures can be used to generate the 5 ^z -cap of a nucleic acid molecule, such as a polynucleotide which functions as an mRNA molecule.

[00064] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wild-type or physiological) 5^Z -caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e. non-enzymatically) or enzymatically synthesized and/or linked to the polynucleotides of the invention.

[00065] For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5^Z -5^Z -triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3 ^z -O-methyl group (i.e., N7,3 ' -O-dimethyl-guanosine-5 ^z -triphosphate-5 ' - guanosine (m7G-3^z mppp-G; which may equivalently be designated 3^Z O-Me-m7G(5^z )ppp(5 ^z )G). The 3^Z -0 atom of the other, unmodified, guanine becomes linked to the 5^Z -terminal nucleotide of the capped polynucleotide. The N7- and 3^Z -O-methlyated guanine provides the terminal moiety of the capped polynucleotide.

[00066] Another example of a cap is mCAP, which is similar to ARCA but has a 2^Z -O- methyl group on guanosine (i.e., N7,2 ' -O-dimethyl-guanosine-5 ' -triphosphate-5 ^z - guanosine, m7Gm-ppp-G).

[00067] Viral Sequences. Additional viral sequences such as, but not limited to, the translation enhancer sequence of the barley yellow dwarf virus (BYDV-PAV), the Jaagsiekte sheep retrovirus (JSRV) and/or the Enzootic nasal tumor virus (See e.g., International Pub. No. WO2012129648; herein incorporated by reference in its entirety) can be engineered and inserted in the polynucleotides of the invention and can stimulate the translation of the construct in vitro and in vivo. Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day 7 post-transfection. [00068] IRES Sequences. Further, provided are polynucleotides (e.g., peptide-encoding polynucleotides featured in the NAVs of the invention) which may contain an internal ribosome entry site (IRES). First identified as a feature Picoma virus RNA, IRES plays an important role in initiating protein synthesis in absence of the 5^Z cap structure. An IRES may act as the sole ribosome binding site or may serve as one of multiple ribosome binding sites of an mRNA. Polynucleotides containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes (“multicistronic nucleic acid molecules”). When polynucleotides are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the invention include without limitation, those from coxsackievirus B3 (CVB3), picomaviruses (e g., FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).

[00069] Poly-A Tails. During RNA processing, a long chain of adenine nucleotides (poly-A tail) may be added to a polynucleotide such as an mRNA molecule in order to increase stability. Immediately after transcription, the 3^Z end of the transcript may be cleaved to free a 3^Z hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long.

[00070] According to the present invention, terminal groups on the poly A tail may be incorporated for stabilization into polynucleotides of the invention (e.g., peptide-encoding polynucleotides featured in the RNAVs of the invention). Polynucleotides of the present invention may include des-3^z hydroxyl tails. They may also include structural moieties or 2^Z - Omethyl modifications as taught by Junjie Li, et al. (Current Biology, Vol. 15, 1501-1507, Aug. 23, 2005, the contents of which are incorporated herein by reference in its entirety).

[00071] The polynucleotides may be designed to encode transcripts with alternative poly A tail structures including histone mRNA. These mRNAs are distinguished by their lack of a 3^Z poly(A) tail, the function of which is instead assumed by a stable stem-loop structure and its cognate stem-loop binding protein (SLBP); the latter carries out the same functions as those of PABP on polyadenylated mRNAs

[00072] Unique poly-A tail lengths provide certain advantages to the polynucleotides of the present invention (e.g., peptide-encoding polynucleotides featured in the NAVs of the invention).

[00073] Generally, the length of a poly-A tail, when present, is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the polynucleotide or region thereof includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1.500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1.000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

[00074] In one embodiment, the poly-A tail is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide. This design may be based on the length of a coding region, the length of a particular feature or region or based on the length of the ultimate product expressed from the polynucleotides.

[00075] In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or feature thereof. The poly-A tail may also be designed as a fraction of the polynucleotides to which it belongs. In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the poly-A tail. Further, engineered binding sites and conjugation of polynucleotides for Poly-A binding protein may enhance expression. [00076] Start Codon Region. In some aspects the polynucleotides may have regions that are analogous to or function like a start codon region.

[00077] In one embodiment, the translation of a polynucleotide may initiate on a codon which is not the start codon AUG. Translation of the polynucleotide may initiate on an alternative start codon such as, but not limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU, TTG/UUG. As a non-limiting example, the translation of a polynucleotide begins on the alternative start codon ACG. As another non-limiting example, polynucleotide translation begins on the alternative start codon CTG or CUG. As yet another non-limiting example, the translation of a polynucleotide begins on the alternative start codon GTG or GUG.

[00078] Nucleotides flanking a codon that initiates translation such as, but not limited to, a start codon or an alternative start codon, are known to affect the translation efficiency, the length and/or the structure of the polynucleotide. (See e.g., Matsuda and Mauro PLoS ONE, 2010 5: 11; the contents of which are herein incorporated by reference in its entirety). Masking any of the nucleotides flanking a codon that initiates translation may be used to alter the position of translation initiation, translation efficiency, length and/or structure of a polynucleotide.

[00079] Stop Codon Region. In one aspect, the polynucleotides may include at least one or two stop codons before the 3^Z untranslated region (UTR). The stop codon may be selected from TGA, TAA and TAG. In one aspect, the polynucleotides include the stop codon TGA and one additional stop codon. In a further embodiment the addition stop codon may be TAA. In another embodiment, the polynucleotides of the present invention include three stop codons.

[00080] Signal Sequences. The polynucleotides described herein may also encode additional features which facilitate trafficking of the polypeptides to therapeutically relevant sites. One such feature which aids in protein trafficking is the signal sequence. As used herein, a “signal sequence” or “signal peptide” is a polynucleotide or polypeptide, respectively, which is from about 9 to 200 nucleotides (3-60 amino acids) in length which is incorporated at the 5^Z (or N- terminus) of the coding region or polypeptide encoded, respectively. Addition of these sequences result in trafficking of the encoded polypeptide to the endoplasmic reticulum through one or more secretory pathways. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported. [00081] Protein Cleavage Signals and Sites. In certain aspects, polypeptides of the invention (e.g., peptide polypeptides) may include various protein cleavage signals and/or sites.

[00082] In one embodiment, the polypeptides of the present invention may include at least one protein cleavage signal containing at least one protein cleavage site. The protein cleavage site may be located at the N-terminus, the C-terminus, at any space between the N- and the C-termini such as, but not limited to, half-way between the N- and C-termini, between the N-terminus and the half-way point, between the half-way point and the C-terminus, and combinations thereof.

[00083] In one embodiment, the polynucleotides of the present invention may be engineered such that the polynucleotide contains at least one encoded protein cleavage signal. The encoded protein cleavage signal may be located in any region including but not limited to before the start codon, after the start codon, before the coding region, within the coding region such as, but not limited to, half way in the coding region, between the start codon and the half way point, between the half way point and the stop codon, after the coding region, before the stop codon, between two stop codons, after the stop codon and combinations thereof.

[00084] In one embodiment, the polynucleotides of the present invention may include at least one encoded protein cleavage signal containing at least one protein cleavage site. The encoded protein cleavage signal may include, but is not limited to, signalase cleavage signal, a proprotein convertase (or prohormone convertase), thrombin and/or Factor Xa protein cleavage signal.

[00085] Codon Optimization. The polynucleotides contained in the NAVs of the invention, their regions or parts or subregions may be codon optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove/add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art, nonlimiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods. In one embodiment, the ORF sequence is optimized using optimization algorithms.

[00086] In some embodiments, a 5^Z UTR and/or a 3^Z UTR region may be provided as flanking regions. Multiple 5^Z or 3^Z UTRs may be included in the flanking regions and may be the same or of different sequences. Any portion of the flanking regions, including none, may be codon optimized and any may independently contain one or more different structural or chemical modifications, before and/or after codon optimization.

[00087] In Vitro Transcription-Enzymatic Synthesis. cDNA encoding the polynucleotides described herein may be transcribed using an in vitro transcription (IVT) system. The system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as, but not limited to, polymerases able to incorporate polynucleotides (e.g., modified nucleic acids).

[00088] Solid-Phase Chemical Synthesis. Chimeric polynucleotides or circular polynucleotides described herein may be manufactured in whole or in part using solid phase techniques.

[00089] Solid-phase chemical synthesis of polynucleotides or nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Impurities and excess reagents are washed away and no purification is required after each step. The automation of the process is amenable on a computer-controlled solid-phase synthesizer. Solid-phase synthesis allows rapid production of polynucleotides or nucleic acids in a relatively large scale that leads to the commercial availability of some polynucleotides or nucleic acids. Furthermore, it is useful in site-specific introduction of chemical modifications in the polynucleotide or nucleic acid sequences. It is an indispensable tool in designing modified derivatives of natural nucleic acids.

[00090] Liquid Phase Chemical Synthesis. The synthesis of chimeric polynucleotides or circular polynucleotides of the present invention (e.g., peptide-encoding polynucleotides featured in the NAVs of the invention) by the sequential addition of monomer building blocks may be carried out in a liquid phase. A covalent bond is formed between the monomers or between a terminal functional group of the growing chain and an incoming monomer. Functional groups not involved in the reaction must be temporarily protected. After the addition of each monomer building block, the reaction mixture has to be purified before adding the next monomer building block. The functional group at one terminal of the chain has to be deprotected to be able to react with the next monomer building blocks. A liquid phase synthesis is labor- and time-consuming and cannot not be automated. Despite the limitations, liquid phase synthesis is still useful in preparing short polynucleotides in a large scale. Because the system is homogenous, it does not require a large excess of reagents and is cost-effective in this respect.

[00091] Combination of Synthetic Methods. The synthetic methods discussed above each has its own advantages and limitations. Attempts have been conducted to combine these methods to overcome the limitations. Such combinations of methods are within the scope of the present invention.

III. Nucleic Acid Modifications

[00092] In certain embodiments, polynucleotides described herein can include various substitutions and/or insertions. As used herein the terms “chemical modification” or, as appropriate, “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribo- or deoxyribonucleosides in one or more of their position, pattern, percent or population. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5^Z -terminal mRNA cap moieties. In a polypeptide, the term “modification” refers to a modification as compared to the canonical set of 20 amino acids. [00093] The modifications may be various distinct modifications. In some embodiments, the regions may contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified polynucleotide, introduced to a cell may exhibit reduced degradation in the cell, as compared to an unmodified polynucleotide.

[00094] The polynucleotides of the NAs of the invention can include any useful modification, such as to the sugar, the nucleobase, or the intemucleoside linkage (e.g. to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications according to the present invention may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.

[00095] Non-natural modified nucleotides may be introduced to polynucleotides during synthesis or post-synthesis of the chains to achieve desired functions or properties. The modifications may be on intemucleotide lineage, the purine or pyrimidine bases, or sugar. The modification may be introduced at the terminal of a chain or anywhere else in the chain; with chemical synthesis or with a polymerase enzyme.

[00096] Modified Polynucleotide Molecules. The present invention also includes building blocks, e.g., modified ribonucleosides, and modified ribonucleotides, of polynucleotide molecules, e.g., of the NAVs of the invention. For example, these building blocks can be useful for preparing the polynucleotides of the invention.

[00097] Modifications on the Sugar. The modified nucleosides and nucleotides which may be incorporated into a polynucleotide can be modified on the sugar of the ribonucleic acid. For example, the 2^Z hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2^Z -position include, but are not limited to, H, halo, optionally substituted Cl-6 alkyl: optionally substituted Cl -6 alkoxy; optionally substituted C6- 10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted C6-10 aryloxy; optionally substituted C6-10 aryl-Cl-6 alkoxy, optionally substituted Cl-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), — O(CH2CH2O)nCH2CH2R, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20); locked” nucleic acids (LNA) in which the 2^Z -hydroxyl is connected by a Cl -6 alkylene or Cl- 6 heteroalkylene bridge to the 4^Z -carbon of the same ribose sugar, where exemplary bridges included methylene, propylene, ether, or amino bridges; aminoalkyl, as defined herein; aminoalkoxy, as defined herein; amino as defined herein; and amino acid, as defined herein

[00098] Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary, non-limiting modified nucleotides include replacement of the oxygen in ribose (e.g., with S, Se. or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphorami date backbone); multicyclic forms (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replace with a -L- threofuranosyl-(3

and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone). The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a polynucleotide molecule can include nucleotides containing, e.g., arabinose, as the sugar. Such sugar modifications are taught International Application Number PCT/2012/058519 filed Oct. 3, 2012 (Attorney Docket Number M9) and U.S. Provisional Application No. 61/837,297 filed Jun. 20, 2013 (Attorney Docket Number M36) the contents of each of which are incorporated herein by reference in its entirety. [00099] Modifications on the Nucleobase. As described herein “nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). As described herein, “nucleotide” is defined as a nucleoside including a phosphate group. The modified nucleotides may by synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleosides). The polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphoester linkages, in which case the polynucleotides would comprise regions of nucleotides.

[000100] The modified nucleotide base pairing encompasses not only the standard adenosinethymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil.

[000101] The modified nucleosides and nucleotides can include a modified nucleobase. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. Examples of nucleobase found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine. Such modified nucleobases (including the distinctions between naturally occurring and non-naturally occurring) are taught in International Application Number PCT/2012/058519 filed Oct. 3, 2012 (Attorney Docket Number M9) and U.S. Provisional Application No. 61/837,297 filed Jun. 20, 2013 (Attorney Docket Number M36) the contents of each of which are incorporated herein by reference in its entirety.

IV. Thrombin Derivative Peptides and Thrombin Peptide TP508

[000102] TP508 represents a regenerative portion of human thrombin that is released from dissolving blood clots at sites of injury. This portion of thrombin stimulates regeneration of tissue. TP508 has been shown to: (i) stimulate revascularization and restoration of tissue repair in multiple tissues; (ii) protect, recruit, and stimulate proliferation of progenitor stem cells at sites of injury; (iii) modulate immune responses; (iv) restore nitric oxide (NO)-dependent endothelial function; (v) prevent apoptosis; and (vi) mitigate effects of radiation to prevent multiple organ failure and increase survival.

[000103] It has been discovered that TP508 prevents TNFa-induced permeability of human pulmonary endothelial cells in vitro. TP508 also counteracts the proinflammatory effects of TNFa on endothelial cells and monocytes, thus serving to reverse pathological inflammatory responses. Described below are methods and composition for the use of a nucleic acid (e.g., mRNA) encoding TP508 to reduce the progression of ARDS.

[000104] Thrombin peptide derivatives (also: "thrombin derivative peptides") are analogs of thrombin that have an amino acid sequence derived at least in part from that of thrombin and are active at the non-proteolytically activated thrombin receptor (NPAR). Thrombin peptide derivatives can include, for example, peptides that are produced by recombinant DNA methods and peptides produced synthetically, which can comprise amino acid substitutions compared to thrombin and/or modified amino acids, especially at one or both termini.

[000105] Thrombin peptide derivatives of the present invention include thrombin derivative peptides described in U.S. Patents 5,352,664 and 5,500,412, each of which is incorporated herein by reference in their entirety. In one embodiment, the thrombin peptide derivatives of the present invention are a physiologically functional equivalent, i.e., a polypeptide with no more than about fifty amino acids, preferably no more than about thirty amino acids and having sufficient homology to the fragment of human thrombin. Certain aspects include a nucleic acid of gcgggctataaaccggatgaaggcaaacgcggcgatgcgtgcgaaggcgatagcggcggcccgtttgtg (SEQ ID NO: 1) encoding Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly- Gly-Pro-Phe-Val (SEQ ID NO:2; TP508).

[000106] In one embodiment, the serine esterase conserved sequence comprises the nucleic acid sequence of SEQ ID NO:3 (tgcgaaggcgatagcggcggcccgtttgtg) encoding SEQ ID NO:4 (Cys- Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val). It is understood, however, that zero, one, two or three amino acids in the serine esterase conserved sequence can differ from the corresponding amino acid in SEQ ID NO:4. Preferably, the amino acids in the serine esterase conserved sequence can differ by 1, 2, 3, or 4 conservative substitutions as defined below, and are more preferably highly conservative substitutions.

[000107] In another embodiment, the serine esterase conserved sequence comprises the nucleic acid sequence of SEQ ID NO: 5 (tgcgaaggcgatagcggcggcccgtttgtg) encoding the amino acid sequence Cys-Xl-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val (SEQ ID NO:6); XI is Glu or Gin and X2 is Phe, Met, Leu, His or Vai). In a preferred embodiment, the thrombin peptide derivative comprises a serine esterase conserved sequence and a polypeptide having a more specific thrombin amino acid sequence Arg-Gly-Asp-Ala. One example of a thrombin peptide derivative of this type is encoded by SEQ ID NO:7 (tgcgaaggcgatagcggcggcccgtttgtg) encoding Arg-Gly- Asp-Ala-Cys-Xl-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val (SEQ ID NO: 8). XI and X2 are as defined above. The nucleic acid encoding a thrombin peptide derivative can comprise the nucleic acid sequence gcgggctataaaccggatgaaggcaaacgcggcgatgcgtgcgaaggcgatagcggcggcccgtttgtg SEQ ID NOV encoding the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp- Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 10), provided that zero, one, two or three amino acids at positions 1-9 in the thrombin peptide derivative differ. Preferably, the amino acid residues in the thrombin peptide derivative which differ are conservative substitutions as defined below, and are more preferably highly conservative substitutions.

[000108] A preferred thrombin peptide derivative for use in the disclosed method is encoded by gcgggctataaaccggatgaaggcaaacgcggcgatgcgtgcgaaggcgatagcggcggcccgtttgtg (SEQ ID NO: 11) comprising the amino acid sequence Ala-Gly-Tyr-Lys-Pro-Asp-Glu-GIy-Lys-Arg-Gly-Asp-Ala- Cys-Xl-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val (SEQ ID NO: 12). Another preferred thrombin peptide derivative for use in the disclosed method is encoded by gcgggctataaaccggatgaaggcaaacgcggcgatgcgtgcgaaggcgatagcggcggcccgtttgtg (SEQ ID NO: 13) comprising the amino acid sequence of Asp-Asn-Met- Phe-Cys-Ala-Gly-Tyr-Lys-Pro-Asp-Glu- Gly-Lys-Arg-Gly-Asp-Ala-Cys-Xl-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val-Met-Lys-Ser-Pro-Phe (SEQ ID NO: 14), wherein XI is Glu or Gin; X2 is Phe, Met, Leu, His or Vai.

[000109] TP508 is an example of a thrombin peptide derivative and is 23 amino acid residues long, wherein the N-terminal amino acid residue Ala is unsubstituted and the COOH of the C- terminal amino acid Vai is modified to an amide represented by -C(O)NH2. Another example of a thrombin peptide derivative comprises the amino acid sequence where both N- and C-termini are unsubstituted ("deamide TP508"). Other examples of thrombin peptide derivatives which can be used in the disclosed method include N-terminal truncated fragments of TP508 (or deamide TP508), the N-terminal truncated fragments having at least fourteen amino acids, or C-terminal truncated fragments of TP508 (or deamide TP508), the C-terminal truncated fragments having at least eighteen amino acids.

[000110] As used herein, a "conservative substitution" in a polypeptide or peptide is the replacement of an amino acid with another amino acid that has the same net electronic charge and approximately the same size and shape. Amino acids with aliphatic or substituted aliphatic amino acid side chains have approximately the same size when the total number of carbon and heteroatoms in their side chains differs by no more than about four. They have approximately the same shape when the number of branches in their side chains differs by no more than one. Amino acids with phenyl or substituted phenyl groups in their side chains are considered to have about the same size and shape. Listed below are five groups of amino acids. Replacing an amino acid in a polypeptide or peptide with another amino acid from the same group results in a conservative substitution:

[000111] Group I: glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, and non-naturally occurring amino acids with C1-C4 aliphatic or C1-C4 hydroxyl substituted aliphatic side chains (straight chained or monobranched).

[000112] Group II: glutamic acid, aspartic acid and non-naturally occurring amino acids with carboxylic acid substituted C1-C4 aliphatic side chains (unbranched or one branch point).

[000113] Group III: lysine, ornithine, arginine and non-naturally occurring amino acids with amine or guanidino substituted C1-C4 aliphatic side chains (unbranched or one branch point).

[000114] Group IV: glutamine, asparagine and non-naturally occurring amino acids with amide substituted Cl -C4 aliphatic side chains (unbranched or one branch point).

[000115] Group V: phenylalanine, phenylglycine, tyrosine and tryptophan. [0001 16] As used herein, a "highly conservative substitution" in a polypeptide is the replacement of an amino acid with another amino acid that has the same functional group in the side chain and nearly the same size and shape. Amino acids with aliphatic or substituted aliphatic amino acid side chains have nearly the same size when the total number of carbon and heteroatoms in their side chains differs by no more than two. They have nearly the same shape when they have the same number of branches in their side chains. Examples of highly conservative substitutions include valine for leucine, threonine for serine, aspartic acid for glutamic acid and phenylglycine for phenylalanine. Examples of substitutions which are not highly conservative include alanine for valine, alanine for serine and aspartic acid for serine.

[000117] In one embodiment of the invention, the thrombin peptide derivatives are modified relative to the thrombin peptide derivatives described above, wherein cysteine residues of thrombin peptide derivatives are replaced with amino acids having similar size and charge properties to minimize dimerization of the peptides. Examples of suitable amino acids include alanine, glycine, serine, or an S' -protected cysteine. Preferably, cysteine is replaced with alanine. The modified thrombin peptide derivatives have about the same biological activity as the unmodified thrombin peptide derivatives. See Publication No. US 2005/0158301 Al, which is hereby incorporated by reference.

[0001 18] "Treating" means that following a period of administering the thrombin peptide derivative or composition comprising a thrombin peptide derivative, a beneficial therapeutic and/or prophylactic result is achieved, which can include a decrease in the severity of symptoms or delay in or inhibition of the onset of symptoms, increased longevity and/or more rapid or more complete resolution of the disease or condition, or other improved clinical outcome as measured according to the site that is being observed or the parameters measured for a particular disease or disorder. "Reducing the risk" refers to decreasing the probability of developing a disease, disorder or medical condition, in a subject, wherein the subject is, for example, a subject who is at risk for developing the disease, disorder or condition.

V. Pharmaceutical Compositions

[000119] The present invention provides pharmaceutical compositions including NAs and NA compositions and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients. The present invention provides NAs and NA pharmaceutical compositions and complexes optionally in combination with one or more pharmaceutically acceptable excipients. Pharmaceutical compositions may optionally comprise one or more additional active substances, e.g., therapeutically and/or prophylactically active substances. Pharmaceutical compositions of the present invention may be sterile and/or pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21^z ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[000120] In some embodiments, compositions are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the NAs or the polynucleotides contained therein, e.g., peptide-encoding polynucleotides, for example, RNA polynucleotides, to be delivered as described herein.

[000121] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.

[000122] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit. [000123] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%. e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

[000124] Formulations. The NAs of the invention can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein (peptide) in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients of the present invention can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with NAVs (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.

[000125] Accordingly, the formulations of the invention can include one or more excipients, each in an amount that may increases the stability of the NAV, increases cell transfection by the NAV, increases the expression of polynucleotides encoded protein, and/or alters the release profde of polynucleotide encoded proteins. Further, the polynucleotides of the present invention may be formulated using self-assembled nucleic acid nanoparticles.

[000126] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.

[000127] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[000128] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

[000129] In some embodiments, the formulations described herein may contain at least one polynucleotide, e.g., peptide-encoding polynucleotide. As a non-limiting example, the formulations may contain 1, 2, 3, 4 or 5 polynucleotides.

[000130] In one embodiment, the formulations described herein may comprise more than one type of polynucleotide, e.g., peptide-encoding polynucleotide. In one embodiment, the formulation may comprise a chimeric polynucleotide in linear and circular form. In another embodiment, the formulation may comprise a circular polynucleotide and an IVT polynucleotide. In yet another embodiment, the formulation may comprise an IVT polynucleotide, a chimeric polynucleotide and a circular polynucleotide.

[000131] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component s) of the pharmaceutical composition.

[000132] In some embodiments, the particle size of the lipid nanoparticle may be increased and/or decreased. The change in particle size may be able to help counter biological reaction such as, but not limited to, inflammation or may increase the biological effect of the modified mRNA delivered to mammals.

[000133] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, surface active agents and/or emulsifiers, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in the pharmaceutical formulations of the invention.

[000134] Liposomes, Lipoplexes, and Lipid Nanoparticles

[000135] The NAs of the invention can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, pharmaceutical compositions of NAs include liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.

[000136] In one embodiment, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from l,2-dioleyloxy-N,N- dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), l,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l ,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by reference in its entirety) and liposomes which may deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

[000137] As an example a liposome can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15% 1 ,2-di oleyl oxy-N,N- dimethylaminopropane (DODMA), as described by Jeffs et al. As another example, certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be l,2-distearloxy-N,N- dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or l,2-dilinolenyloxy-3- dimethylaminopropane (DLenDMA), as described by Heyes et al.

[000138] Peptides and Proteins. The NAs of the invention can be formulated with peptides and/or proteins in order to increase transfection of cells by the polynucleotide. In one embodiment, peptides such as, but not limited to, cell penetrating peptides and proteins and peptides that enable intracellular delivery may be used to deliver pharmaceutical formulations. A non-limiting example of a cell penetrating peptide which may be used with the pharmaceutical formulations of the present invention includes a cell-penetrating peptide sequence attached to polycations that facilitates delivery to the intracellular space, e.g., HIV-derived TAT peptide, penetratins, transportans, or hCT derived cell-penetrating peptides (see, e.g., Caron et al., Mol. Ther. 3(3):310-8 (2001); Langel, Cell-Penetrating Peptides: Processes and Applications (CRC Press, Boca Raton Fla., 2002); El-Andaloussi et al., Curr. Pharm. Des. 1(28):3597-611 (2003); and Deshayes et al., Cell. Mol. Life Sci. 62(16): 1839-49 (2005), all of which are incorporated herein by reference in their entirety). The compositions can also be formulated to include a cell penetrating agent, e.g., liposomes, which enhance delivery of the compositions to the intracellular space. NAVs of the invention may be complexed to peptides and/or proteins such as, but not limited to, peptides and/or proteins from Aileron Therapeutics (Cambridge, Mass.) and Permeon Biologies (Cambridge, Mass.) in order to enable intracellular delivery (Cronican et al., ACS Chem. Biol. 2010 5:747-752; McNaughton et al., Proc. Natl. Acad. Sci. USA 2009 106:6111-6116; Sawyer, Chem Biol Drug Des. 2009 73:3-6; Verdine and Hilinski, Methods Enzymol. 2012; 503:3-33; all of which are herein incorporated by reference in its entirety). [000139] Cells. The NAs of the invention can be transfected ex vivo into cells, which are subsequently transplanted into a subject.

[000140] Suspension Formulations. In some embodiments, suspension formulations are provided comprising NAs, water immiscible oil depots, surfactants and/or co-surfactants and/or co-solvents. Combinations of oils and surfactants may enable suspension formulation with NAs. Delivery of NAs in a water immiscible depot may be used to improve bioavailability through sustained release of NAs from the depot to the surrounding physiologic environment and prevent polynucleotides degradation by nucleases.

[000141] In some embodiments, suspension formulations of NA may be prepared using combinations of polynucleotides, oil-based solutions and surfactants. Such formulations may be prepared as a two-part system comprising an aqueous phase comprising polynucleotides and an oil-based phase comprising oil and surfactants. Exemplary oils for suspension formulations may include, but are not limited to sesame oil and Miglyol (comprising esters of saturated coconut and palmkernel oil-derived caprylic and capric fatty acids and glycerin or propylene glycol), com oil, soybean oil, peanut oil, beeswax and/or palm seed oil. Exemplary surfactants may include, but are not limited to Cremophor, polysorbate 20, polysorbate 80, polyethylene glycol, transcutol, Capmul®, labrasol, isopropyl myristate, and/or Span 80. In some embodiments, suspensions may comprise co-solvents including, but not limited to ethanol, glycerol and/or propylene glycol.

[000142] Excipients. NA pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, flavoring agents, stabilizers, antioxidants, osmolality adjusting agents. pH adjusting agents and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

[000143] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions. The composition may also include excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents.

[000144] Cryoprotectants. In some embodiments, NA formulations may comprise cyroprot ectants. As used herein, there term “cryoprotectant” refers to one or more agent that when combined with a given substance, helps to reduce or eliminate damage to that substance that occurs upon freezing. In some embodiments, cryoprotectants are combined with NAs in order to stabilize them during freezing. Frozen storage of NAs between -20° C. and -80° C. may be advantageous for long term (e g., 36 months) stability of polynucleotide. In some embodiments, cryoprotectants are included in NAV formulations to stabilize polynucleotide through freeze/thaw cycles and under frozen storage conditions. Cryoprotectants of the present invention may include, but are not limited to sucrose, trehalose, lactose, glycerol, dextrose, raffinose and/or mannitol. Trehalose is listed by the Food and Drug Administration as being generally regarded as safe (GRAS) and is commonly used in commercial pharmaceutical formulations.

[000145] Bulking Agents. In some embodiments, NA formulations may comprise bulking agents. As used herein, there term “bulking agent” refers to one or more agents included in formulations to impart a desired consistency to the formulation and/or stabilization of formulation components. In some embodiments, bulking agents are included in lyophilized NA formulations to yield a “pharmaceutically elegant” cake, stabilizing the lyophilized NAs during long term (e.g. 36 month) storage. Bulking agents of the present invention may include, but are not limited to sucrose, trehalose, mannitol, glycine, lactose and/or raffinose. In some embodiments, combinations of cryoprotectants and bulking agents (for example, sucrose/glycine or trehalose/mannitol) may be included to both stabilize NAs during freezing and provide a bulking agent for lyophilization.

[000146] Administration. The NAs of the present invention may be administered by any route which results in a therapeutically effective outcome.

[000147] Parenteral and Injectable Administration

[000148] Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.

[000149] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents. Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[000150] Dosing. The present invention provides methods comprising administering NAs and in accordance with the invention to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. Compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[000151] In certain embodiments, compositions in accordance with the present invention may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect (see e.g., the range of unit doses described in International Publication No WO2013078199, herein incorporated by reference in its entirety). The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used.

[000152] According to the present invention, NAs may be administered in split-dose regimens. As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g., two or more administrations of the single unit dose. As used herein, a “single unit dose” is a dose of any therapeutic administer in one dose/at one time/single route/single point of contact, i.e., single administration event. As used herein, a “total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose. In one embodiment, the NAs of the present invention are administered to a subject in split doses. The NAVs may be formulated in buffer only or in a formulation described herein. [000153] Multi-Dose and Repeat-Dose Administration

[000154] In some embodiments, NA compounds and/or compositions of the present invention may be administered in two or more doses (referred to herein as “multi-dose administration”). Such doses may comprise the same components or may comprise components not included in a previous dose. Such doses may comprise the same mass and/or volume of components or an altered mass and/or volume of components in comparison to a previous dose. In some embodiments, multi-dose administration may comprise repeat-dose administration. As used herein, the term “repeat-dose administration” refers to two or more doses administered consecutively or within a regimen of repeat doses comprising substantially the same components provided at substantially the same mass and/or volume. In some embodiments, subjects may display a repeat-dose response. As used herein, the term “repeat-dose response” refers to a response in a subject to a repeat-dose that differs from that of another dose administered within a repeat-dose administration regimen. In some embodiments, such a response may be the expression of a protein in response to a repeat-dose comprising NAV. In such embodiments, protein expression may be elevated in comparison to another dose administered within a repeatdose administration regimen or protein expression may be reduced in comparison to another dose administered within a repeat-dose administration regimen. Alteration of protein expression may be from about 1% to about 20%, from about 5% to about 50% from about 10% to about 60%, from about 25% to about 75%, from about 40% to about 100% and/or at least 100%. A reduction in expression of mRNA administered as part of a repeat-dose regimen, wherein the level of protein translated from the administered RNA is reduced by more than 40% in comparison to another dose within the repeat-dose regimen is referred to herein as “repeat-dose resistance.”

VI. Methods of Treating

[000155] The compositions described herein can be used to express TP508 or its analogs in various tissues to treat various pathologic conditions. Non-limiting examples of such conditions are provided below.

[000156] Chronic and Acute Pulmonary Conditions. Pulmonary fibrosis (PF) is a progressive respiratory disorder characterized by a scarring and thickening of the lining of the lungs that causes irreversible loss of ability to transport and exchange oxygen. As lung tissue scars, it becomes more rigid, making it more difficult for the lungs to inflate and deflate. When this happens, less oxygen is transferred into the bloodstream, making it harder to breathe. As PF worsens, a person becomes progressively weaker and short of breath, and this damage eventually results in death. When an etiology for PF cannot be clearly identified, the condition is termed idiopathic pulmonary fibrosis (IPF). Certain aspects of the present invention are directed to a method for treating pulmonary fibrosis (PF) and idiopathic pulmonary fibrosis (IPF). Although the cause of PF and IPF may be different, the signs and symptoms of PF and IPF are the same, and the present invention is effective to treat PF and IPF, regardless the cause of the disease. The method comprises the step of administering an effective amount of a engineered mRNA construct to a patient who is suffering from pulmonary fibrosis. Expression of TP508 or its analogs can ameliorate fibrosis in the alveoli of a patient and treat pulmonary fibrosis. The treatment can also reduce disease complications, such as lung inflammation, shortness of breath, pain crisis, pneumonia, and increase survival. In one embodiment, the methods can treat pulmonary fibrosis in a subject with chronic obstructive pulmonary disease (COPD), with pulmonary arterial hypertension (PAH), with sickle cell disease (SCD), with scleroderma or with lung cancer.

[000157] Acute Respiratory Distress Syndrome (ARDS). Acute respiratory distress syndrome (“ARDS”) is a manifestation of a systemic inflammatory response that develops, for example, as a direct or indirect consequence of lung injury. The hallmark of ARDS is deterioration in blood oxygenation and respiratory system compliance because of permeability edema. A consensus definition of ARDS distinguishes ARDS from other conditions such as acute lung injury (ALI) based on differing severity of clinical lung injury: patients with less severe hypoxemia are considered to have ALI, and those with more severe hypoxemia are considered to have the ARDS. ARDS is defined by the following criteria (Bernard et al., Am. J. Respir. Crit. Care Med 149, 818-824, 1994): 1. Acute onset; 2. Bilateral infiltrates on chest radiography; 3. Pulmonary- artery wedge pressure is less than or equal to 18 mm Hg or the absence of clinical evidence of left atrial hypertension; and 4. hypoxemia, as determined by the ratio of partial pressure of arterial oxygen to fraction of inspired oxygen, i.e., PaC^FiCL, is less than or equal to 200.

[000158] ARDS is often progressive, characterized by distinct stages exhibiting different clinical, histopathological and radiographic parameters. An acute phase of ARDS involves acute neutrophil influx to the lungs e.g., arising from e.g., sepsis, pneumonia, aspiration, ischemia (circulatory arrest, hemorrhagic shock), trauma, severe asthma, poisoning, severe acute respiratory syndrome (SARS), influenza, or infection. The acute phase of ARDS is characterized by rapid onset of respiratory failure in a patient having a predisposition for the condition, especially arterial hypoxemia that is refractory to oxygen supplementation. Broncho-alveolar- lavage (BAL) studies reveal substantial inflammation in areas that appear normal by radiography or tomography as well as in areas that exhibit alveolar filling, consolidation, and atelectasis. Pathologically, the lung in this acute phase exhibits diffuse alveolar damage, with neutrophils, macrophages, erythrocytes, hyaline membranes, capillary injury, and disruption of the alveolar epithelium.

[000159] The acute phase may progress to fibrosing alveolitis with persistent hypoxemia, increased alveolar dead space and further decrease in alveolar compliance. In patients with ARDS the microvascular, interstitial, and alveolar spaces of the lungs are the primary targets for fibrin deposition, and micro thrombus formation can occur in multiple organs, with lungs and kidneys as the most exposed, leading to multiple organ failure (MOF). Pulmonary hypertension may arise from obliteration of the pulmonary capillary bed and, in severe cases this may cause right ventricular failure. Pneumothorax may occur in about 10-13% of subjects.

[000160] Certain embodiments are directed to methods of treatment of ARDS or ALT, particularly virus induced ARDS, and/or one or more complications thereof or for the prophylactic treatment of one or more clinical disorders associated with the development of ARDS or ALI. Complications include neutrophilic inflammation and its downstream consequences such as, for example, alveolar filling, alveolar epithelial damage or loss, amongst others, and prophylactic treatment of a subject having one or more risk factors for ARDS but that has not yet suffered an acute phase of ARDS or a complication thereof. In this respect, it will be evident that the reduction of neutrophilic inflammation and enhancement/induction of alveolar re-epithelialization are more pertinent to therapeutic regimens, and that the prevention of neutrophilic inflammation and/or the prevention or reduction of alveolar epithelial injury or loss are more pertinent to prophylactic regimens. In certain aspects, the method includes administering to a subject in need thereof a formulation comprising an mRNA engineered to express TP508 or an analog or derivative thereof at a location and for a time and under conditions sufficient to reduce or prevent ARDS, ALI, or related pathology.

[000161] There is often a delay between a precipitating factor e.g., trauma, poisoning, viral infection, etc. and the onset of ARDS or ALI, which the inventors reason provides a window of opportunity for administering a formulation of the invention or other formulation comprising an mRNA engineered to express TP508 or an analog or derivative thereof. Accordingly, in one example, this invention provides a method for the prophylaxis or prevention of ARDS comprising administering to a subject at risk of developing ARDS or exposed to one or more risk factors of ARDS (e.g., infection by a respiratory virus such as SARS-CoV-2) a formulation comprising an mRNA engineered to express TP508 or an analog or derivative thereof. In certain aspects, the subject is suffering from breathing difficulty and/or has reduced breathing capability. In certain aspects, the subject can inhale the formulation and the formulation is administered to the subject by inhalation. In another example, the formulation is administered by injection. The formulation can be administered to the subject by injection via an intravenous, intraperitoneal, intramuscular, or subcutaneous routes.

[000162] Brain Cancers. The method includes contacting a brain cancer, e.g., medulloblastoma cancer cell, with an effective amount of a thrombin peptide expressed from an mRNA engineered to express TP508 or an analog or derivative thereof. In certain aspects the brain cancer is exposed to a cancer treatment such as radiation, chemotherapy, or a combination thereof. The cancer cells may be cancer stem cells. The cancer cells may be ex vivo or in vivo. As used herein, “ex vivo” refers to a cell that has been removed from the body of an animal. Ex vivo cells include, for instance, primary cells (e.g., cells that have recently been removed from a subject and are capable of limited growth in tissue culture medium), and cultured cells (e.g., cells that are capable of long-term culture in tissue culture medium). As used herein, “in vivo” refers to a cell that is present within the body of an animal. The use of a thrombin peptide sensitizes a cancer cell, such as cancer stem cell, to a cancer treatment such as radiation. Thus, the method can result in decreased viability of a cancer cell, reduced ability of a cancer stem cell to repair DNA damage from radiation exposure, down-regulated activation of NF-kB in a cancer cell, or a combination thereof. Certain embodiments are directed to methods of sensitizing a cell to radiation (radiation sensitization or radiosensitization), such as a cancer cell. [000163] In one embodiment, the method includes administering to the subject an effective amount of an mRNA engineered to express TP508 or an analog or derivative thereof, and exposing cells to a therapeutic cancer treatment, such as radiation, chemotherapy, or a combination thereof. In one embodiment, the methods disclosed herein include treating one or more symptoms or clinical signs of brain cancer, e.g., medulloblastoma, in a subject. As used herein, the term “symptom” refers to subjective evidence of a brain cancer experienced by the subject and caused by the cancer. As used herein, the term “clinical sign” or, simply, “sign” refers to objective evidence caused by the cancer. Symptoms and/or clinical signs associated with medulloblastoma and the evaluations of such symptoms and/or clinical signs are routine and known in the art. The use of a thrombin peptide sensitizes the cancer cells, such as cancer stem cells, to a cancer treatment such as radiation, chemotherapy, or a combination thereof. Thus, the method can result in decreased viability of cancer cells of the tumor, such as cancer stem cells, increased remission of the medulloblastoma, decreased tumor growth, increased tumor shrinkage, decreased frequency of metastasis, decreased tumor relapse, increased cancer survival rate of the subject, or a combination thereof, compared to the subject exposed to the therapeutic cancer treatment and not administered the thrombin peptide derivative. Remission of the brain cancer may be partial (e.g., decrease or disappearance of some symptoms or signs) or complete (e.g., decrease or disappearance of and symptoms and signs). The subject may have been treated previously, for instance, the subject may have been in remission.

[000164] Dermal Conditions. Dermal skin ulcers refer to lesions on the skin caused by superficial loss of tissue that fail to heal normally due to defects in healing processes, vascular insufficiency or pressure. Dermal skin ulcers which can be treated by the method of the present invention include decubitus ulcers, diabetic ulcers, venous stasis ulcers, and arterial ulcers. Decubitus wounds refer to chronic ulcers that result from pressure applied to areas of the skin for extended periods of time. Wounds of this type are often called bedsores or pressure sores. Venous stasis ulcers result from the stagnation of blood or other fluids from defective veins. Arterial ulcers refer to necrotic skin in the area around arteries having poor blood flow.

[000165] Compounds that stimulate or activate the non-proteolytically activated thrombin receptor (hereinafter “NPAR”) promote or stimulate healing of chronic dermal skin ulcers. Compounds which stimulate NPAR are said to be NPAR agonists. NPAR is a high-affinity thrombin receptor present on the surface of most cells. This NPAR component is largely responsible for high-affinity binding of thrombin, proteolytically inactivated thrombin, and thrombin derived peptides to cells. NPAR mediates a number of cellular signals that are initiated by thrombin independent of its proteolytic activity. An example of one such signal is the upregulation of annexin V and other molecules identified by subtractive hybridization (see Sower, et. al., Experimental Cell Research 247:422 (1999)). NPAR is therefore characterized by its high affinity interaction with thrombin at cell surfaces and its activation by proteolytically inactive derivatives of thrombin and thrombin derived peptide agonists.

[000166] In one embodiment, the method includes administering to a subject suffering from a dermal condition an effective amount of an mRNA engineered to express TP508 or an analog or derivative thereof.

[000167] Vascular and Cardiovascular Conditions. Cardiovascular diseases are generally characterized by an impaired supply of blood to the heart or other target organs. Myocardial infarction (MI) results from narrowed or blocked coronary arteries in the heart which starves the heart of nutrients and oxygen. When the supply of blood to the heart is compromised, cells respond by generating compounds that induce the growth of new blood vessels to increase the supply of blood to the heart. These new blood vessels are called collateral blood vessels. The process by which new blood vessels are induced to grow out of the existing vasculature is termed angiogenesis, and the substances that are produced by cells to induce angiogenesis are the angiogenic factors.

[000168] When heart muscle is deprived of oxygen and nutrients due to vascular occlusion, the heart muscle tissue becomes ischemic and loses its ability to contract and function. This loss of function may be restored by natural signals from the ischemic heart muscle that induce angiogenic revascularization through development of collateral vessels that bypass the occlusion. This revascularization or angiogenesis involves the stimulation of endothelial cell proliferation and migration and budding off of new blood vessels. In many cases, however, the natural signals are not sufficient to cause collateral vessel growth and the ischemic tissue can become fibrotic or necrotic. If this process is not reversed by procedures to open the occluded vessels or further induction of collateral blood vessels, the heart may become totally dysfunctional and require transplantation.

[000169] The compositions and formulations described herein can be employed to induce angiogenic proliferation and migration of endothelial cells resulting in formation of new capillaries and collateral vessels to help restore function to damaged or ischemic heart tissue. These compositions and formulations may be directly injected into or applied to heart tissue during open chest procedures for bypass surgery or insertion of ventricular assist devices or delivered by catheter injection into the heart as an mRNA engineered to express TP508 or an analog or derivative thereof.

[000170] Endothelial cell proliferation, such as that which occurs in angiogenesis, is also useful in preventing or inhibiting restenosis following balloon angioplasty. The balloon angioplasty procedure often injures the endothelial cells lining the inner walls of blood vessels and disrupts the integrity of the vessel wall. Smooth muscle cells and inflammatory cells often infiltrate into the injured blood vessels causing a secondary obstruction in a process known as restenosis. Stimulation of the proliferation and migration of the endothelial cells located at the periphery of the balloon-induced damaged area to cover the luminal surface of the vessel with a new monolayer of endothelial cells would potentially restore the original structure of the blood vessel.

[000171] Certain aspects are directed to methods of treating endothelial dysfunction in a subject, wherein the subject is suffering from one or more diseases or conditions selected from the group consisting of hypertension, congestive heart failure, coronary artery disease, stroke, cerebrovascular disease, peripheral vascular disease, diabetes, erectile dysfunction, atherosclerosis, asthma, rheumatoid arthritis, pulmonary hypertension, acute lung injury, chronic obstructive pulmonary disease (COPD), cystic fibrosis, inflammatory lung disease, hyperhomocysteinemia, sickle cell disease, pre-eclampsia, chronic renal failure, chronic renal dysfunction, renal microvascular disease, hepatic reperfusion injury, neuropathy, Alzheimer's disease, thyroid disease, sepsis, thrombosis, multiple organ failure, inflammatory bowel disease, and radiation damage.

[000172] The present invention is also directed to methods of treating endothelial dysfunction at a site in a subject in need of treatment, comprising administering to the subject a an mRNA engineered to express TP508 or an analog or derivative thereof in a therapeutically effective amount. The method can include administering a combination therapy comprising one or more NPAR agonists and one or more angiogenic growth factors.

[000173] Endothelialization can be re-endothelialization after angioplasty, to reduce, inhibit or prevent restenosis. Those of skill in the art will recognize that patients treated according to the methods of the present invention may be treated with or without a stent.

[000174] An inflatable balloon catheter with an mRNA engineered to express TP508 or an analog or derivative thereof coating the balloon or a catheter that expresses the peptide locally to or in the wall of the vessel may also be employed to deliver the substance to a targeted artery. The mRNA engineered to express TP508 or an analog or derivative thereof described herein can be employed to induce proliferation and migration of the endothelial cells located at the periphery of the balloon induced damaged area in order to cover the luminal surface of the vessel with a new monolayer of endothelial cells, hoping to restore the original structure of the blood vessel. Coronary angioplasty is frequently accompanied by deployment of an intravascular stent to help maintain vessel function and avoid restenosis. Stents have been coated with heparin to prevent thrombosis until the new channel formed by the stent can endothelialize. The mRNA engineered to express TP508 or an analog or derivative thereof described herein can be locally applied or systemically administered to enhance endothelialization of the vessel or vessel wall and/or to modulate other processes to inhibit or reduce thrombosis and restenosis.

[000175] In one embodiment, the method includes administering to a subject suffering from a vascular or cardiovascular condition an effective amount of an mRNA engineered to express TP508 or an analog or derivative thereof.

[000176] Bone Conditions. “Osteoinduction” refers to stimulating bone growth at a site within a subject at which little or no bone growth would occur if the site were left untreated. Sites which could therapeutically benefit from the induction of bone growth are referred to as “in need of osteoinduction”. Examples include non-union fractures or other severe or massive bone trauma. It is noted that bone growth normally occurs at bone injuries such as simple or hairline fractures and well opposed complex fractures with minimal gaps without the need for further treatment. Such injuries are not considered to be “in need of osteoinduction”. [000177] Simple fracture repair appears to be quite different from the induction of bone formation required to fill non-union fractures, segmental gaps or bone voids caused, for example, by removal of a bone tumor or cyst. These cases require bone grafting or induction of new bone growth generally employing some type of matrix or scaffolding to serve as a bone growth substitute. Induced bone growth can also be therapeutically beneficial at certain sites within a subject (referred to as “ectopic” sites) where bone tissue would not normally be found, such as a site in need of a bone graft or bone fusion. Fusions are commonly used to treat lower back pain by physically coupling one or more vertebrae to its neighbor. The bone created by such a fusion is located at a site not normally occupied by bone tissue. Osteoinduction at these ectopic sites can act as a “graft substitute” whereby induced bone growth between the vertebrae takes the place of a graft and obviates the need for a second operation to harvest bone for the grafting procedure. Induction of bone growth is also needed for treating acquired and congenital craniofacial and other skeletal or dental anomalies (see e.g., Glowacki et al., Lancet 1 : 959 (1981)); performing dental and periodontal reconstructions where lost bone replacement or bone augmentation is required such as in a jaw bone; and supplementing alveolar bone loss resulting from periodontal disease to delay or prevent tooth loss (see e.g., Sigurdsson et al., J. Periodontal., 66: 511(1995)).

[000178] Applicants have discovered that compounds which stimulate or activate the non- proteolytically activated thrombin receptor (hereinafter “NPAR”) are osteoinductive. Such compounds are said to be NPAR agonists. NPAR is a high-affinity thrombin receptor present on the surface of most cells. This NPAR component is largely responsible for high-affinity binding of thrombin, proteolytically inactivated thrombin, and thrombin derived peptides to cells. NPAR mediates cellular signals that are initiated by thrombin independent of its proteolytic activity. NPAR is therefore characterized by its high affinity interaction with thrombin at cell surfaces and its activation by proteolytically inactive derivatives of thrombin and thrombin derived peptide agonists as described below.

[000179] In one embodiment, the method includes administering to a subject suffering from a bone condition an effective amount of an mRNA engineered to express TP508 or an analog or derivative thereof. [000180] Connective Tissue Conditions. Sites in need of cartilage growth, repair or regeneration are found in subjects with osteoarthritis. Osteoarthritis or degenerative joint disease is a slowly progressive, irreversible, often monoarticular disease characterized by pain and loss of function. The underlying cause of the pain and debilitation is cartilage degradation that is one of the major symptoms of the disease. Hyaline cartilage is a flexible tissue that covers the ends of bones and lies between joints such as the knee. It is also found in between the bones along the spine. Cartilage is smooth, allowing stable, flexible movement with minimal friction, but is also resistant to compression and able to distribute applied loads. As osteoarthritis progresses, surfaces of cartilage and exposed underlying bone become irregular. Instead of gliding smoothly, boney joint surfaces rub against each other, resulting in stiffness and pain. Regeneration of damaged cartilage and the growth of new cartilage at these arthritic sites would relieve the pain and restore the loss of function associated with osteoarthritis.

[000181] Cartilage damage can also occur from trauma resulting from injury or surgery. Sports injuries are a common cause of cartilage damage, particularly to joints such as the knee. Traumatic injury to cartilage can result in the same type of functional impairment. Therefore, sites in a subject with cartilage that has been damaged by trauma or disease are in need of treatment to restore or promote the growth of cartilage.

[000182] Applicants have discovered that compounds which stimulate or activate the non- proteolytically activated thrombin receptor (hereinafter “NPAR”) can stimulate chondrocytes to proliferate. Chondrocytes are cells which make up about 1% of the volume of cartilage and which replace degraded matrix molecules to maintain the correct volume and mechanical properties of the tissue. Applicants have also found that compounds which stimulate or activate NPAR stimulate proteoglycan synthesis in chondrocytes. Proteoglycan is a major cartilage component. Based on these results, Applicants can deliver an NPAR agonist via an mRNA engineered to express TP508 or an analog or derivative thereof. TP508 was administered to defects in rabbit trochlear grove cartilage and the peptide stimulated repair of the defect that included formation of new cartilage with a normal cartilage surface. The peptide also stimulated layering and integration of this new cartilage into adjacent, uninjured cartilage and restoration of the subchondral bone. It is concluded that NPAR agonists can induce cartilage growth and repair when administered to sites needing cartilage growth and/or repair. Sustained localized expression from an mRNA engineered to express TP508 or an analog or derivative thereof can provide TP508 for treatment of these conditions.

[000183] In one embodiment, the method includes administering to a subject suffering from a connective tissue condition an effective amount of an mRNA engineered to express TP508 or an analog or derivative thereof.

[000184] Degenerative Conditions. Agonists of a non-proteolytically activated receptor (NPAR) can be used in methods for treating a disease or disorder in a subject by administering to the subject a therapeutically effective amount of an mRNA engineered to express TP508 or an analog or derivative thereof., wherein the disease or disorder is scleroderma, macular degeneration, diabetic retinopathy, Huntington's disease, Parkinson's disease, closed head trauma, glaucoma, optic neuritis or allograft vasculopathy. NPAR agonists may exert their effect by inhibiting apoptosis. NPAR agonists can be thrombin peptide derivatives encoded by an mRNA engineered to express TP508 or an analog or derivative thereof.

[000185] One or more NPAR agonists, and in particular, one or more thrombin peptide derivatives, can be used in methods to treat any of the following: scleroderma, macular degeneration, diabetic retinopathy, Huntington's disease, Parkinson's disease, closed head trauma, glaucoma, optic neuritis, and allograft vasculopathy. Compositions comprising NPAR agonists can be administered to a subject in need of treatment of the diseases and disorders described herein. Treatment can ameliorate the disease or disorder, or alleviate the symptoms thereof. NPAR agonists can be administered to subjects who can benefit from therapeutic intervention causing complete or partial alleviation of symptoms. NPAR agonists can be administered to subjects, (e.g., human patients) at risk for developing a disorder described herein, to reduce the probability of developing the disorder. For example, treatment can cause a reduction in the probability of developing the disorder by up to 20, 30, 40, 50, 60, 70, 80, or 90 percent. Treatment can in some cases, delay the development of a disorder, reduce symptoms, or delay severity of symptoms.

[000186] In one embodiment, the method includes administering to a subject suffering from a degenerative condition an effective amount of an mRNA engineered to express TP508 or an analog or derivative thereof. [000187] As used herein the term “treatment” includes therapeutic treatment of a subject who has already suffered from or is suffering from a condition, e.g., ARDS etc. or a complication thereof. Consistent with this construction, the term “prevent” or “prevention” as used throughout this specification shall not be taken to require an absolute i.e., 100% abrogation of a condition, and it is sufficient that there is a significant reduction in these adverse consequences of a condition using the method and formulations of the present invention compared to the absence of treatment in accordance with the present invention. Similarly, the term “reduction” or “reduce” as used throughout this specification shall not be taken to require an abrogation of a condition or symptoms in a subject more than a significant effect compared to the absence of treatment in accordance with the present invention. Similarly, the terms “enhance”, “enhancement”, “induce” and “induction” as used throughout this specification shall not be taken to require any particular quantitative change, merely an improvement that is significant compared to the absence of treatment in accordance with the present invention.

[000188] As used herein, the term “administer” shall be taken to mean that a formulation is applied in a location directly or indirectly in a subject/patient, e.g., applied to the respiratory system of a subject including the nasal passage, buccal cavity, throat or esophagus or lung, by inhalation and/or applied to the circulatory system of a subject by injection intramuscularly, subcutaneously, intravenously, intraperitoneally etc., including single or repeated or multiple dosages by any administration route. As used herein the term “inhalation” shall be taken to include “aspiration”. In certain aspect administration of TP508 or an analog or derivative thereof includes administration of an mRNA that can in turn express TP508 or an analog or derivative thereof under appropriate conditions.

[000189] As used herein, the term “subject in need thereof’ shall be taken to mean a subject that has developed or suffers from a condition or one or more complications thereof or is predisposed by virtue of having one or more risk factors to suffering from such condition or one or more complications thereof. In one example, a subject has not yet suffered significant impairment of breathing or significant damage to the alveolar epithelium and has one or more risk factors for ARDS or acute lung injury or a complication thereof, such as diagnosis of a respiratory virus infection. [000190] The present invention clearly contemplates repeated administration of a formulation as described herein according to any embodiment in the therapy or prophylaxis of ARDS and complications thereof. For example, repeated injection and/or inhalation of a formulation of the present invention may be required to reduce or prevent inflammatory responses in the lung for a long period of time, e.g., during sepsis or persistent or long-term infection by a bacterial agent or virus.

[000191] Repeated administration of a formulation as described herein may be timed to ensure a sufficiently high concentration of the bioactive peptide component of the formulation in plasma of the subject and/or at the site of action in the treatment regimen. For example, second and/or subsequent doses of a formulation of the invention as described according to any embodiment hereof may be administered at a time when serum or tissue concentration of a peptide provided by one or more previous doses fall(s) below a desired level at which it is active or provides sufficient benefit to the patient. Such booster doses of a formulation of the present invention are clearly contemplated in the prophylaxis and/or therapy of ARDS and/or one or more complications thereof according to the present invention.

[000192] For example, the present invention provides a method of treatment or prophylaxis of a subject in need thereof, said method comprising:

[000193] (i) identifying a subject suffering from a condition, e.g., ARDS, and/or one or more complications thereof or one or more clinical disorders associated with the development of of a condition, e.g., ARDS, or is at risk of suffering from a condition, e.g., ARDS, and/or one or more complications thereof or one or more clinical disorders associated with the development of a condition, e.g., ARDS; and (ii) administering a formulation as described herein to the subject.

VII. Kits and Devices

[000194] The invention provides a variety of kits for conveniently and/or effectively carrying out methods of the present invention. Typically kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments. [000195] In one aspect, the present invention provides kits comprising the NA molecules (including any proteins or polynucleotides) of the invention. In one embodiment, the kit comprises one or more functional peptides or function fragments thereof.

[000196] The kits can be for protein production, comprising a first polynucleotides comprising a translatable region of a peptide. The kit may further comprise packaging and instructions and/or a delivery agent to form a formulation composition. The delivery agent may comprise a saline, a buffered solution, or a delivery agent.

[000197] In one embodiment, the buffer solution may include sodium chloride, calcium chloride, phosphate and/or EDTA. In another embodiment, the buffer solution may include, but is not limited to, saline, saline with 2 mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% Mannitol, 5% Mannitol with 2 mM calcium, Ringer's lactate, sodium chloride, sodium chloride with 2 mM calcium and mannose. In a further embodiment, the buffer solutions may be precipitated or it may be lyophilized. The amount of each component may be varied to enable consistent, reproducible higher concentration saline or simple buffer formulations. The components may also be varied in order to increase the stability of polynucleotides in the buffer solution over a period of time and/or under a variety of conditions.

[000198] In one aspect, the present invention provides kits for protein production, comprising: a polynucleotide comprising a translatable region, provided in an amount effective to produce a desired amount of a protein encoded by the translatable region when introduced into a target cell.

[000199] Devices. The present invention provides for devices which may incorporate RNAs comprising polynucleotides that encode polypeptides of interest, e.g., encode peptide(s). These devices contain in a stable formulation the reagents to synthesize a polynucleotide in a formulation available to be immediately delivered to a subject in need thereof, such as a human patient.

[000200] Devices for administration may be employed to deliver the NAs of the present invention according to single, multi- or split-dosing regimens taught herein.

[000201] Method and devices known in the art for multi-administration to cells, organs and tissues are contemplated for use in conjunction with the methods and compositions disclosed herein as embodiments of the present invention. These include, for example, those methods and devices having multiple needles, hybrid devices employing for example lumens or catheters as well as devices utilizing heat, electric current or radiation driven mechanisms.

VIII. Examples

[000202] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, considering the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

CONSTRUCTION OF FLAG-TP508

[000203] Cloning strategy and design. The shuttle vector pESP-2 was purchased from Stratagene, Inc. (La Jolla, CA). The vector contains a GST (glutathione S transferase) sequence containing the FLAG peptide (DYKDDDDK). The multiple cloning site is immediately downstream (3’) of the FLAG peptide sequence, which is the recognition target for enterokinase (cleavage after the lysine residue, K). To ensure that we could purify TP508 devoid of any additional amino acids, it was necessary to construct a minigene which overlapped the FLAG sequence and the multiple cloning site. The designed minigene contains a 5’ BamH I site, the FLAG peptide (exactly as it is in the vector), the TP508 sequences (derived from gi: 5922005; nucleotides 1681 to 1749), a stop codon immediately following the TP508 sequences (TA A), and a 3’ Bgl II restriction enzyme site. The BamH I/Bgl II restricted minigene would then be cloned into a similarly restricted vector.

[000204] Cloning of the vector. The oligonucleotides were ordered from Integrated DNA Technologies, Inc. (Coralville, IA). The single-stranded oligonucleotides were annealed and then digested with BamH I and Bgl II, using manufacturer’s recommended protocol (Promega, Madison, WI). The pESP2 vector was similarly digested with the same enzymes. The digested double-stranded oligonucleotide (minigene) and vector were ligated at 16°C overnight. Three microliters of the ligation mixture were transformed into DH5a E. coli.

[000205] Analysis of the clones. Transformed bacterial colonies were selected using ampicillin. Sixty colonies were amplified, and DNA minipreparations obtained using available kits (GenElute™ Plasmid Miniprep Kit, Sigma Chemical Co., St. Louis, MO; Quantum Prep Plasmid Miniprep, Bio-Rad Laboratories, Hercules, CA). The plasmid DNA was digested with Nhe I, which is present in the vector, but should be absent in vectors containing the FLAG-TP508 minigene insert. Clones 7, 11, 17, 41, 44, 47, 48, 54 and 56 were identified as potentially having the minigene insert.

[000206] Sequence verification of the clones. DNA from these clones was then sequenced by the Protein Chemistry Core Laboratory at UTMB. Clones 41, 47 and 54 were found to have the desired sequence; clones 11 and 56 had two tandem TP508 monomers. In both clones the first TP508 monomer is in frame and the second monomer is out of frame, with respect to the GST- FLAG sequence. Unfortunately, both have a stop codon between the TP508 monomers, rendering them essentially TP508 monomers at the protein level, like clones 41, 47, and 54.

[000207] In Vitro Transcription. A linearized DNA plasmid template can be used for in vitro transcription. The in vitro transcribed mRNA contains the cap, 5^Z and 3^Z untranslated regions (UTRs), the open reading frame (ORF) and the poly(A) tail (FIG. 1), which determine the translational activity and stability of the mRNA molecule after its transfer into cells.

[000208] In Vivo Translation. mRNA is endocytosed by cell-specific mechanisms and enters endosomal pathways. mRNA is released into the cytoplasm. mRNA is translated by the protein synthesis machinery of host cells. Translation is terminated by degradation of mRNAs by exonucleases.

EXAMPLE 2

CONSTRUCTIONS OF GFP-TP508

[000209] Cloning strategy and design. The plasmid pEGFP-C2 was purchased from Clontech (Palo Alto, CA), under license agreement with Aurora Biosciences, Inc., on the condition that it be used for non-commercial purposes. It consists of the enhanced green fluorescent protein under the regulation of the CMV promoter, followed by a multiple cloning site and an SV40 polyadenylation signal. Oligonucleotides were designed to incorporate the nucleic acid sequence for human TP508 (derived from gi: 5922005; nucleotides 1681 to 1749) with artificial EcoR I and Bam H I restriction enzyme sites on the 5’ and 3’ ends of the oligonucleotide, respectively. This construction strategy allows for the introduction of multiple TP508 sequences in tandem using another pair of oligonucleotides incorporating the human TP508 sequences flanked by BamH I site on the 5’ end and EcoR I site on the 3’ end.

[000210] Cloning of the vector. The oligonucleotides were ordered from Integrated DNA Technologies, Inc. (Coralville, IA). The single- stranded oligonucleotides were boiled for 5 minutes and annealed at room temperature. The annealed double-stranded oligonucleotide (minigene) was then digested with EcoR I and BamH I, using manufacturer’s recommended protocol (Promega, Madison, WI). The pEGFP-C2 vector was similarly digested with the same enzymes. The digested minigene and vector were ligated at 16°C overnight. Three microliters of the ligation mixture were transformed into DH5a E. coli.

[000211] Analysis of the clones. Transformed bacterial colonies were selected using kanamycin (30pg/ml). Thirteen colonies were amplified, and DNA minipreparations obtained using available kits (GenElute™ Plasmid Miniprep Kit, Sigma Chemical Co., St. Louis, MO; Quantum Prep Plasmid Miniprep, Bio-Rad Laboratories, Hercules, CA). The plasmid DNA was digested with Kpn I, which is present in the vector, but should be absent in vectors containing the TP508 minigene insert. All clones, except clones 4 and 9, were identified as potentially having the TP508 insert.

[000212] Sequence verification of the clones. DNA from the positive clones (1-3, 5-8, 10-16) was then sequenced by the Protein Chemistry Core Laboratory at UTMB. Clonel 1 was found to have the desired sequence.

[000213] In Vitro Transcription. A linearized DNA plasmid template can be used for in vitro transcription. The in vitro transcribed mRNA contains the cap, 5^Z and 3^Z untranslated regions (UTRs), the open reading frame (ORF) and the poly(A) tail (FIG. 1), which determine the translational activity and stability of the mRNA molecule after its transfer into cells. [000214] In Vivo Translation. mRNA is endocytosed by cell-specific mechanisms and enters endosomal pathways. mRNA is released into the cytoplasm. mRNA is translated by the protein synthesis machinery of host cells. Translation is terminated by degradation of mRNAs by exonucleases.

Claims

1. An engineered messenger ribonucleic acid (mRNA) comprising an open reading frame encoding a TP508 peptide.

2. The engineered mRNA of claim 1, wherein the mRNA is linear.

3. The engineered mRNA of claim 2, further comprising a 5’ UTR.

4. The engineered mRNA, further comprising a 3’ UTR.

5. The engineered mRNA, further comprising a polyadenylation segment.

6. The engineered mRNA, further comprising 5’ region comprising from 5’ to 3’ (i) a 5’ external homology segment, (ii) a 3’ intron and exon segment, (iii) a 5’ internal homology segment, and (iv) a poly adenosine/cytosine spacer.

7. The engineered mRNA, further comprising 3’ region comprising from 5’ to 3’ (i) a poly adenosine/cytosine spacer, (ii) a 3’ internal homology segment, (iii) a 5’ intron and exon segment, and (iv) a 3 ’ external homology segment.

8. The engineered mRNA, wherein the ORF has a nucleotide sequence that is 80, 85, 90, 95, 98, 99, 100 % identical to SEQ ID NO: 1.

9. A DNA construct encoding the engineered mRNA of claim 1.

10 A method of treating a subject with acute respiratory distress or a subject at risk of developing acute respiratory distress comprising administering to the subject an engineered mRNA of claim 1.

11. The method of claim 10, wherein the formulation is administered to the subject by injection, inhalation, or instillation.

12. The method of claim 10, wherein the formulation is administered to the subject by intravenous (IV) injection.

13. The method of claim 10, wherein the formulation is administered to the subject by subcutaneous injection.

14. The method of claim 10, wherein the formulation is administered to the subject by inhalation or instillation.

15. The method of claim 10, wherein the subject is diagnosed with a viral infection.

16. The method of claim 10, wherein the subject is diagnosed with a respiratory virus infection.

17. The method of claim 10, wherein the subject is diagnosed with a coronavirus or influenza infection.

18. The method of claim 10, wherein the subject is diagnosed with a SARS-CoV-2 infection.

19. A method for ameliorating post-respiratory virus infection syndrome comprising administering to the subject of an engineered mRNA of claim 1.

20. The method of claim 19, wherein the formulation is administered to the subject by injection, inhalation, or instillation.

21. The method of claim 19, wherein the formulation is administered to the subject by intravenous (IV) injection.

22. The method of claim 19, wherein the formulation is administered to the subject by subcutaneous injection.

23. The method of claim 19, wherein the formulation is administered to the subject by inhalation or instillation.

24. The method of claim 19, wherein the respiratory virus infection is a coronavirus or influenza infection.

25. The method of claim 19, wherein the coronavirus infection is a SARS-CoV-2 infection.