Polynucleotide stuffer sequences for AAV vectors

Abstract

This present disclosure provides a stuffer sequence for optimal packaging and productivity of a recombinant adeno-associated virus (rAAV) vector. This present disclosure also provides methods of designing, manufacturing, and using such stuffer sequence.

Description

PATENT Attorney Docket No.: 079445-015110PC-1515381 Client Reference No. S24-338 POLYNUCLEOTIDE STUFFER SEQUENCES FOR AAV VECTORS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 691,148, filed September 5, 2024, the disclosure of which is herein incorporated by reference in its entirety for all purposes. BACKGROUND

[0002] Maximum packaging capacity of single stranded(ss) recombinant adeno associated virus (rAAV) used in therapy is ~ 4.6 kb. An ideal payload size of recombinant AAV for optimal packaging and productivity is between 4-4.6 kb.

[0003] A payload size less than 4 kb compromises productivity and a payload size of less than 2.4 kb compromises genome integrity that produces truncated products. Potential consequences of non-optimal therapeutic ss rAAV payload size often generate heterogenous drug product, causing safety, efficacy, and manufacturing concerns. For examples, production may have higher percentages of truncated genomes (safety and efficacy concern), and non- specific payload including carry-over from packaging plasmids and host DNA (safety concern), and reduced titers (manufacturing concern). As therapeutic AAV payloads may not reach the optimal size (4-4.6 kb), a DNA stuffer sequence is often needed for size adjustment. SUMMARY

[0004] In one aspect, the present disclosure provides a recombinant polynucleotide comprising a first nucleic acid sequence flanked by two inverted terminal repeat (ITR) sequences, wherein the first nucleic acid sequence encodes a 3' untranslated region (3'UTR) of a bone morphogenetic protein 10 (BMP-10) transcript. In some embodiments, the BMP-10 transcript is a human BMP-10 transcript. In some embodiments, the length of the recombinant polynucleotide is between about 3-5 kb, optionally between about 4-4.7 kb. In some embodiments, the first nucleic acid sequence comprises a sequence at least 80% identity to SEQ ID NO: 1. In some embodiments, the two ITR sequences are 5’ ITR comprising a sequence at least 80% identity to SEQ ID NO: 2 and 3’ ITR comprising a sequence at least80% identity to SEQ ID NO: 3. In some embodiments, the recombinant polynucleotide comprises a nucleic acid sequence at least 80% identity to SEQ ID NO: 4.

[0005] In some embodiments, at least a portion of the first nucleic acid sequence is replaced with a second nucleic acid sequence, and wherein the length of the recombinant polynucleotide keeps the same. In some embodiments, the second nucleic acid sequence encodes a protein of interest operably linked to a promoter.

[0006] In some embodiments, a vector comprises the recombinant polynucleotide disclosed here. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV serotype 8 (AAV8) vector or an AAV serotype 9 (AAV9) vector. The present disclosure also provides a pharmaceutical composition comprising a recombinant polynucleotide or a vector described herein, and a pharmaceutically acceptable carrier.

[0007] In another aspect, the present disclosure provides a method of preventing or treating a disease in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein.

[0008] In another aspect, the present disclosure provides a method of manufacturing the recombinant polynucleotide described herein. In some embodiments, the method comprises culturing a host cell comprising a rAAV vector, wherein the rAAV vector comprises the recombinant polynucleotide, under conditions suitable for rAAV production. In some embodiments, the rAAV vector is a self-complementary AAV (scAAV) vector. In some embodiments, the scAAV vector is a scAAV serotype 8 (scAAV8) vector or a scAAV serotype 9 (scAAV9) vector. In some embodiments, the rAAV vector is a single-stranded AAV (ssAAV) vector. In some embodiments, the ssAAV vector is a ssAAV serotype 8 (ssAAV8) vector or a ssAAV serotype 9 (ssAAV9) vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG.1 illustrates a 4611 bp sequence from the 3’UTR of a human BMP-10 transcript that can be used as a stuffer for AAV payload design and manufacturing.

[0010] FIG. 2 illustrates safety and manufacturability evaluation of the BMP10-3’UTR payload using ML / AI by FormBio. A. The BMP10-3’UTR payload. B. Truncation map of the BMP10-3’UTR payload. Truncation estimate along the length of the construct with the annotations (x-axis), and the modeled sequence truncation propensity (y-axis). Replicationforward sequence (top plot) and reverse (bottom plot). C. 2° & 3° Structure locations of the BMP10-3’UTR payload. Probability of a 2° or 3° structure (y-axis) and annotations and bp (x- axis). Probability of a 2° and 3° structure motif (green line). Darker green shaded areas are hot spots for high correlation between a predicted truncation event and a 2° or 3° structure element. D. GC content & CpG islands of the BMP10-3’UTR payload. Percent CpG islands identified (orange line) along the length of the CDS. Percent GC content (blue line). The shaded regions in orange indicate where we identified the highest likelihood of CpG island formation. E. Truncation map of a sub-optimal construct. F. 2° & 3° Structure locations of a sub-optimal construct. G. GC content & CpG islands of a sub-optimal construct.

[0011] FIG. 3 illustrates safety and manufacturability evaluation of a payload for gene augmentation therapy using ML / AI by FormBio. A. The payload designed for gene augmentation therapy. B. Truncation map of the designed payload. Truncation estimate along the length of the construct with the annotations (x-axis), and the modeled sequence truncation propensity (y-axis). Replication forward sequence (top plot) and reverse (bottom plot). C. 2° & 3° Structure locations of the designed payload. Probability of a 2° or 3° structure (y-axis) and annotations and bp (x-axis). Probability of a 2° and 3° structure motif (green line). Darker green shaded areas are hot spots for high correlation between a predicted truncation event and a 2° or 3° structure element. D. GC content & CpG islands of the designed payload. Percent CpG islands identified (orange line) along the length of the CDS. Percent GC content (blue line). The shaded regions in orange indicate where we identified the highest likelihood of CpG island formation. E. Truncation map of a sub-optimal construct. F.2° & 3° Structure locations of a sub-optimal construct. G. GC content & CpG islands of a sub-optimal construct.

[0012] FIG. 4 illustrates safety and manufacturability evaluation of a payload for gene silencing therapy using ML / AI by FormBio. A. The payload designed for gene silencing therapy. B. Truncation map of the designed payload. Truncation estimate along the length of the construct with the annotations (x-axis), and the modeled sequence truncation propensity (y- axis). Replication forward sequence (top plot) and reverse (bottom plot). C.2° & 3° Structure locations of the designed payload. Probability of a 2° or 3° structure (y-axis) and annotations and bp (x-axis). Probability of a 2° and 3° structure motif (green line). Darker green shaded areas are hot spots for high correlation between a predicted truncation event and a 2° or 3° structure element. D. GC content & CpG islands of the designed payload. Percent CpG islands identified (orange line) along the length of the CDS. Percent GC content (blue line). The shaded regions in orange indicate where we identified the highest likelihood of CpG island formation.E. Truncation map of a sub-optimal construct. F.2° & 3° Structure locations of a sub-optimal construct. G. GC content & CpG islands of a sub-optimal construct.

[0013] FIG. 5 illustrates alkaline gel analysis of the designed payload. A. The unoptimized payload without BMP10-3’UTR stuffer sequence. B. The payload designed for gene augmentation therapy optimized with the BMP10-3’UTR stuffer sequence. C. Alkaline gel analysis of single stranded AAV8 (ssAAV8) vector . 5E10 vector genomes (vg) of each purified vector (ID1-ID4) was heat denatured and subsequently run on an alkaline gel for 5 h and stained with GelRed.

[0014] FIG. 6 illustrates alkaline gel analysis of the designed payload. A. The unoptimized payload without BMP10-3’UTR stuffer sequence. B. The payload designed for gene silencing therapy optimized with the BMP10-3’UTR stuffer sequence. C. Alkaline gel analysis of single stranded AAV8 (ssAAV8) vector. 5E10 vg of each purified vector (ID1-ID3) was heat denatured and subsequently run on an alkaline gel for 5h and stained with GelRed.

[0015] FIG.7 illustrates alkaline gel analysis of the designed payload using a single stranded AAV9 (ssAAV9) vector. A. Alkaline gel analysis of ssAAV9 vector for gene augmentation therapy. B. Alkaline gel analysis of ssAAV9 vector for gene silencing therapy. In both assays, 1E11 vg of each purified vector was heat denatured and subsequently run on an alkaline gel for 5h and stained with GelRed.

[0016] FIG. 8 illustrates an overview of an exemplary manufacturing process of AAV employed at research scale. DETAILED DESCRIPTION I. INTRODUCTION

[0017] The present disclosure provides a stuffer sequence for optimal packaging and productivity of a recombinant adeno associated virus (rAAV) vector. The key properties of an ideal stuffer DNA sequence for a rAAV includes (i) human origin (safety); (ii) no significant secondary structures that may result in truncated products during AAV packaging (safety, efficacy & manufacturability); (iii) no significant CpGs for any potential host innate immune response (safety & efficacy); (iv) absence of any active genetic elements like promoters, enhancers (safety); and (v) absence of any proto-onco sequences (safety). The present disclosure also provides methods of designing, manufacturing, and using the stuffer sequence,as well as a vector or a composition (e.g., a pharmaceutical composition) comprising the stuffer sequence. II. DEFINITIONS

[0018] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present disclosure. For purposes of the present disclosure, the following terms are defined.

[0019] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.

[0020] As used herein, the terms “about” and “around” indicate a close range around a numerical value when used to modify that specific value. If “X” were the value, for example, “about X” or “around X” would indicate a value from 0.9X to 1.1X, e.g., a value from 0.95X to 1.05X, or a value from 0.98X to 1.02X, or a value from 0.99X to 1.01X. Any reference to “about X” or “around X” specifically indicates at least the values X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.1X, and values within this range.

[0021] The term “recombinant” when used with reference, e.g., to a nucleic acid, protein, vector, or cell, indicates that the nucleic acid, protein vector, or cell, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0022] The terms “polynucleotide” and “nucleic acid” are used interchangeably and as used herein refer to both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. In particular embodiments, a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide, and combinations thereof. The terms also include, but is not limited to, single- and double-strandedforms of DNA. In addition, a polynucleotide, e.g., a cDNA or mRNA, may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. Nucleic acid molecules, e.g., oligonucleotide probes or primers, may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analogue, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). The above term is also intended to include any topological conformation, including single-stranded, double- stranded, partially duplexed, triplex, hairpinned, circular and padlocked conformations. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The term also includes codon-optimized nucleic acids that encode the same polypeptide sequence.

[0023] The term “polynucleotide stuffer” or “stuffer” refers a polynucleotide fragment intentionally inserted into a genetic construct of a vector (e.g., a AAV vector) to optimize the size of the genetic construct. As disclosed herein, a stuffer sequence shall not have any observable or detectable negative impact on vector manufacturability and safety.

[0024] The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0025] The term "promoter," as used herein, refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell. Thus, promoters can include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and / or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, further including an enhancer, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on / off, regulate, modulate, etc.) gene transcription. As disclosed herein, the promoter can be a tissue- specific promoter, such as a cardiac myocyte-specific promoter.

[0026] “Percent sequence identity” or “percent identity” or equivalents used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least a specified level of identity, e.g., at least 50% sequence identity with a reference sequence (e.g., any SEQ ID NO included herein). Alternatively, percent identity can be any integer from 50% to 100%. Some embodiments include at least: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared to a reference sequence using the programs described herein, e.g., BLAST using standard parameters, as described below.

[0027] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0028] A "comparison window", as used herein, includes reference to a segment of any one of the numbers of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection.

[0029] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1977) Nucleic Acids Res.25: 3389-3402,respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site.

[0030] The term "vector" as used herein, refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. As disclosed herein, the vector can be a viral vector. For example, viral vectors can be based on adeno-associated virus (AAV), vaccinia virus, poliovirus, retrovirus, lentivirus, adenovirus, SV40, herpes simplex virus, human immunodeficiency virus, and the like. In some embodiments, viral vectors can be virus- like particles. Other useful expression vectors are known to those of skill in the art, and many are commercially available. The following exemplary vectors are provided by way of example for eukaryotic host cells: pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40. Examples of techniques that may be used to introduce a viral vector into a cell include, but not limited to, viral or bacteriophage infection, transfection, protoplast fusion, lipofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, calcium phosphate precipitation, nanoparticle- mediated nucleic acid delivery, and the like. As disclosed herein, the vector can be an adeno- associated virus (AAV) vector. The term “AAV vector” refers to any vector which comprises or derives from components of AAV and is suitable to infect mammalian cells, preferably human cells. The term AAV vector typically designates an AAV type viral particle or virion comprising at least a nucleic acid molecule encoding a therapeutic protein. As described herein, the AAV vector may be derived from various serotypes or from various genomes.

[0031] By “pharmaceutically acceptable,” it is meant that the excipient is compatible with the other ingredients of the formulation and is not deleterious to the recipient thereof.

[0032] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment,” as used herein, can include treatment resulting in inhibiting the disease, i.e., arresting its development; and relieving the disease, i.e., causing regression of the disease. For example, in the case of dilated cardiomyopathy, a response to treatment can include completeresponse, partial response, stable disease, progressive disease, progression free survival, or overall survival.

[0033] An “effective amount” or “therapeutically effective amount” of an interfering RNA (e.g., siRNA) is an amount sufficient to produce the desired effect, e.g., an inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of an interfering RNA. In particular embodiments, inhibition of expression of a target gene or target sequence is achieved when the value obtained with an interfering RNA relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring the expression of a target gene or target sequence include, but are not limited to, examination of protein or mRNA levels using techniques known to those of skill in the art, such as, e.g., dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.

[0034] As used herein, the term “administering” or “administration” includes any route of introducing or delivering an agent (such as a pharmaceutical composition described herein) to a subject. Administration can be carried out by any route suitable for the delivery of the agent. Thus, delivery routes can include, e.g., oral, intranasal, intravenous, intramuscular, intraperitoneal, intraarticular, intradermal, or subcutaneous.

[0035] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. The dose will vary depending on a number of factors, including frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; the route of administration; and the imaging modality of the detectable moiety (if present). One of skill in the art will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical, and depends on the route of administration. For example, a dosage form can be in a liquid, e.g., a saline solution for injection.

[0036] “Subject,” “patient,” “individual” and like terms are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, dogs, cats, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typicallyrefers to an individual under medical supervision. A patient can be an individual that is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc. III. DESCRIPTION OF THE EMBODIMENTS

[0037] The present disclosure provides a recombinant polynucleotide sequence from the 3’ UTR region of the human BMP10 transcript that can act as a stuffer sequence in an AAV payload. This sequence has been evaluated for their compatibility as a stuffer sequence with respect to AAV payload designing both by machine learning based analysis and wet lab validation. 1. Adeno-associated virus (AAV) Stuffer Sequences

[0038] In one aspect, the present disclosure provides a recombinant polynucleotide (e.g., a AAV stuffer sequence) comprising a first nucleic acid sequence flanked by two inverted terminal repeat (ITR) sequences, wherein the first nucleic acid sequence encodes a 3' untranslated region (3'UTR) of bone morphogenetic protein 10 (BMP-10) transcript. In some embodiments, the BMP-10 transcript is a human BMP-10 transcript. In some embodiments, the length of the recombinant polynucleotide is between about 3-5 kb. In some embodiments, the recombinant polynucleotide is about 3, 3.5, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 kb in length. In some embodiments, the length of the recombinant polynucleotide is between about 4-4.7 kb.

[0039] In some embodiments, the recombinant polynucleotide is about 4.6 kb in length. In some embodiments, the first nucleic acid sequence comprises a sequence at least 80%, 85%, or 90% identical to SEQ ID NO: 1. In some embodiments, the first nucleic acid sequence comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1; or comprises a nucleic acid sequence of SEQ ID NO: 1.

[0040] In some embodiments, the two ITR sequences include a 5’ ITR and a 3’ ITR. In some embodiments, the 5’ ITR comprises a nucleic acid sequence at least 80%, 85%, or 90% identical to SEQ ID NO: 2. In some embodiments, the 5’ ITR comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2; or comprises a nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the 3’ ITR comprises a nucleic acid sequence at least 80%, 85%, or 90% identical to SEQ ID NO: 3. In some embodiments, the 3’ ITR comprises a nucleic acid sequence having at least 95%, 96%, 97%,98%, or 99% identity to SEQ ID NO: 3; or comprises a nucleic acid sequence of SEQ ID NO:

[0041] In some embodiments, the recombinant polynucleotide comprises a nucleic acid sequence at least 80%, 85%, or 90% identical to SEQ ID NO: 4. In some embodiments, the recombinant polynucleotide comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4; or comprises a nucleic acid sequence of SEQ ID NO:

[0042] In some embodiments, at least a portion of the first nucleic acid sequence in the recombinant polynucleotide is replaced with a second nucleic acid sequence. In some embodiments, the replacement does not change the length of the recombinant polynucleotide. As disclosed herein, the second nucleic acid sequence can comprise any nucleic acid sequence of interest, as long as the length of the recombinant polynucleotide remains between about 3-5 kb. In some embodiments, the second nucleic acid sequence encodes a protein of interest operably linked to a promoter.

[0043] In some embodiments, the recombinant polynucleotide can be used for gene augmentation therapy. In some embodiments, the second nucleic acid sequence comprises a gene of interest. In some embodiments, the second nucleic acid sequence further comprises a promoter, wherein the gene of interest is operably linked to the promoter. In some embodiments, the second nucleic acid sequence further comprises a polyA sequence at the 3- end of the gene of interest. (e.g., FIG. 3A). In some embodiments, the recombinant polynucleotide comprising the second nucleic acid sequence has a sequence at least 80%, 85%, or 90% identical to any one of SEQ ID NOs: 5-8. In some embodiments, the recombinant polynucleotide comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 5-8; or comprises a nucleic acid sequence of any one of SEQ ID NOs: 5-8.

[0044] In some embodiments, the recombinant polynucleotide can be used for gene silencing therapy. For Examples, the second nucleic acid sequence can comprise a shRNA guide strand and a shRNA-passenger strand. In some embodiments, the shRNA guide strand and the shRNA-passenger strand are linked through a loop sequence. In some embodiments, the shRNA guide strand – loop - shRNA-passenger strand is flanked by a hsa_miR-5’ flanking sequence and a hsa_miR-3’ flanking sequence (e.g., FIG.4A).2. Adeno-Associated Virus (AAV) Vectors

[0045] The present disclosure also provides a vector comprising the recombinant polynucleotide described herein. Such vector can deliver the recombinant polynucleotide comprising a gene of interest into a cell, thereby expressing the gene of interest for e.g., a gene augmentation therapy or a gene silencing therapy.

[0046] In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector. Adeno-associated virus (AAV) is a dependent parvovirus, of approximately twenty nanometers in size. Like other parvoviruses, AAV is a single-stranded, non-enveloped DNA virus, having a genome of about 5000 nucleotides in length, containing two open reading frames. The left-hand open reading frame codes for the proteins responsible for replication (Rep), while the right-hand open reading frame encodes the structural proteins of the capsid (Cap). The open reading frames are flanked by two ITR sequences, which serve as the origin of replication of the viral genome. Furthermore, the genome also contains a packaging sequence, allowing packaging of the viral genome into an AAV capsid.

[0047] AAV requires co-helper functions (which may be provided e.g. by an adenovirus, or by suitable packaging cells or helper plasmids) to undergo a productive infection in cultured cells. In the absence of such helper functions, the AAV virions essentially enter the cells, migrate to the nucleus as a single-stranded DNA molecule, and integrate into the cell genomes. AAV has a broad host range for infectivity, including human cells, is ubiquitous in humans, and is completely non-pathogenic.

[0048] AAV vectors have been designed, produced and used to mediate gene delivery in human subjects, including for therapeutic purposes. Clinical trials are presently ongoing in various countries using AAV vectors. Typically, AAV vectors for use in gene transfer comprise a replication defective AAV genome lacking functional Rep and Cap coding viral sequences. Such replication defective AAV vectors more preferably lack most or all of the Rep and Cap coding sequences, and essentially retain one or two AAV ITR sequences and a packaging sequence.

[0049] Methods of producing such AAV vectors have been disclosed in the literature, including using packaging cells, auxiliary viruses or plasmids, and / or baculovirus systems (Samulski et al., (1989) J. Virology 63, 3822; Xiao et al., (1998) J. Virology 72, 2224; Inoueet al., (1998) J. Virol. 72, 7024; WO98 / 22607; WO2005 / 072364). It should be noted that several of these methods relate to helper-free AAV production, which is a preferred production method within the scope of the present invention. Methods of producing pseudotyped AAV vectors have also been reported (e.g., WO00 / 28004), as well as various modifications or formulations of AAV vectors, to reduce their immunogenicity upon in vivo administration (see e.g., WO01 / 23001; WO00 / 73316; WO04 / 112727; WO05 / 005610; WO99 / 06562).

[0050] In some embodiments, the AAV vector is a self-complementary AAV (scAAV) vector. For scAAV vectors, both the coding and complementary sequence of the transgene expression cassette are present on each plus-and minus-strand genome. ScAAV vectors are generated by deleting the terminal resolution site (trs) from one of the AAV terminal repeats. These modified vectors, whose replicating genome is half the length of the wild type have the tendency to package DNA dimers (McCarty et al., Gene Therapy, 2003). Briefly, during the replication cycle of AAV, Rep endonuclease nicks the trs to initiate a second DNA replication process generating monomeric genomes. Dimeric genomes of scAAV are generated when Rep fails to nick the trs (McCarty, Molecular Therapy 2008). Replication continues through the ITR to generate a dimeric template which initiates a new round of DNA synthesis, producing a dimeric single-strand genome (dimeric inverted repeat genomes). Both strands are thus packaged into the AAV virion as a single molecule. The two halves of the single-strand DNA molecule can then fold and base pair to form a dsDNA molecule. The above-mentioned articles of McCarty et al. describe in detail the production process of an scAAV and may be followed to obtain such scAAV vector described herein. As disclosed herein, a scAVV vector is engineered from the naturally occurring adeno-associated virus (AAV) as its coding region forms an intra-molecular double-stranded DNA template. As such, upon entering into a host cell, the two complementary halves of scAAV can associate to form one double stranded DNA (dsDNA) for immediate replication and transcription. An scAAV can increase and prolong transgene expression in vitro and in vivo, as well as higher in vivo DNA stability and more effective circularization.

[0051] In some embodiments, the AAV vector is a single-stranded AAV (ssAAV) vector. For ssAAV vectors, the coding sequence and complementary sequence of the transgene expression cassette are on separate strands and are packaged in separate viral capsids.

[0052] AAV vectors may be prepared or derived from various serotypes of AAVs. In a particular embodiment, the AAV vector for use in the present disclosure is derived from ahuman AAV virus. Such a human AAV (capsid and ITR) may be derived from any known serotype, e.g. from any one of serotypes 1-11, preferably from AAV2, AAV4, AAV6, AAV8 and AAV9, more preferably from AAV6, AAV8 and AAV9, even more preferably from AAV8 and AAV9. Specific examples of such AAV vectors are vectors comprising an AAV2-derived genome (a nucleic acid molecule comprising an AAV2-derived ITR and an AAV2-derived packaging sequence, operatively linked to a nucleic acid encoding a therapeutic protein, preferably two AAV2-derived ITR flanking an AAV2-derived packaging sequence and a nucleic acid encoding a therapeutic protein) in an AAV2-derived capsid; vectors comprising an AAV4-derived genome in an AAV4-derived capsid; vectors comprising an AAV6-derived genome in an AAV6-derived capsid; vectors comprising an AAV8-derived genome in an AAV8-derived capsid; vectors comprising an AAV9-derived genome in an AAV9-derived capsid. In some embodiments, the AAV vector is an AAV serotype 8 (AAV8) vector. In some embodiments, the AAV vector is a scAAV8 vector. In some embodiments, the AAV vector is a ssAAV8 vector. In some embodiments, the AAV vector is an AAV9 vector. In some embodiments, the AAV vector is a scAAV9 vector. In some embodiments, the AAV vector is a ssAAV9 vector. 3. Pharmaceutical Compositions

[0053] The present disclosure also provides a composition comprising a recombinant polynucleotide or vector described herein. In some embodiments, the composition is a cell comprising a recombinant polynucleotide or vector described herein

[0054] In some embodiments, the composition is a pharmaceutical composition which further comprises a pharmaceutically acceptable carrier. The pharmaceutical composition may be administered in any suitable form, either as a liquid solution or suspension, as a solid form suitable for solution or suspension in liquid prior to injection, as a gel or as an emulsion. The pharmaceutical composition is typically formulated with any appropriate and pharmaceutically acceptable excipient, carrier, adjuvant, diluent, etc. For injection, the excipient may be a liquid, isotonic solution, buffer, such as sterile and pyrogen-free water or a sterile and pyrogen-free phosphate-buffered saline solution. For inhalation, the excipient may be in particulate form. Methods

[0055] In another aspect, the present disclosure provides a method for preventing or treating a disease in a subject, comprising administering to the subject a therapeutically effectiveamount of the pharmaceutical composition described herein (e.g., rAAV particle comprising a rAAV vector comprising the vector stuffer sequence provided herein and an expression construct comprising heterologous nucleic acid sequence encoding a therapeutic agent). In some embodiments, a subject treated in any of the methods described herein is a mammal (e.g., mouse, rat), preferably a primate (e.g., monkey, chimpanzee), or human.

[0056] In another aspect, the present disclosure provides a method of manufacturing the recombinant polynucleotide (e.g., a AAV stuffer sequence), the vector, or the pharmaceutical composition described herein.

[0057] The present disclosure provides compositions and methods for preventing or treating diseases in a subject. The methods comprise administering to the subject a therapeutically effective amount of a pharmaceutical composition. As disclosed herein, the pharmaceutical composition comprises a recombinant polynucleotide comprising a stuffer DNA sequence disclosed herein or a vector comprising such recombinant polynucleotide.

[0058] As disclosed herein, the administration can be any administration route to introduce the pharmaceutical composition into the subject. In some embodiments, the administration includes, without limitation, oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, subcutaneous, and intradermal. More particularly, the administration includes a systemic injection of the the pharmaceutical composition (e.g., comprising a recombinant AAV vector), such as intramuscular (i.m.), intravascular, i.e. intra- arterial (i.a.) or intravenous (i.v.), intraperitoneal (i.p.), sub-cutaneous or transdermic injections. Peripheral administration also includes oral administration of AAV vectors (WO96 / 40954), delivery using implants (WO01 / 91803), or administration by instillation through the respiratory system (for example by the intranasal route), e.g., using sprays, aerosols or any other appropriate formulations. Most preferred systemic administration includes the systemic injection of the pharmaceutical composition, most preferably via an i.m., i.p., i.a. or i.v. injection. Most preferably, the pharmaceutical composition is administered via an i.v. injection.

[0059] The pharmaceutical composition is typically administered in a “therapeutically effective” amount, i.e., an amount that is sufficient to alleviate (e.g., decrease, reduce) at least one of the symptoms associated with the disease state, or to provide improvement in the condition of the subject. It should be pointed out that repeated administrations may be performed, if required, using either the same or different systemic administration routes (and / orthe same or distinct scAAV serotypes). Alternatively, a single administration of the pharmaceutical composition may also be performed.

[0060] Naturally, the amount of pharmaceutical composition in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0061] The amount of pharmaceutical composition administered will depend upon the ratio of therapeutic composition (e.g., the recombinant polynucleotide or the vector described herein) to carrier, the particular therapeutic composition used, the disease or disorder being treated, the age, weight, and condition of the patient, and the judgment of the clinician. In some embodiments, the pharmaceutical composition can be delivered with high penetrance to the heart by antegrade coronary infusion. In some embodiments, the pharmaceutical composition can be delivered with high penetrance to the heart by intravenous infusion. In some embodiments, the pharmaceutical composition can be delivered to the heart at a dose of 108, 109, 1010, 1011, 1012, 1013, 1014, 1015,1016, or 1017viral genomes (vg) / kg body weight, preferably from about 1011to 1016vg / kg body weight, most preferably from 1012to 1014. In some embodiments, the pharmaceutical composition can be delivered to the heart at a dose of 1x, 2x, 3x, 4x, or 5x 1013vg / kg body weight. As disclosed herein, the range of doses shall not cause toxicity. In some embodiments, one dose of administration can confer days, months, or even years of therapeutic benefit. IV. EXAMPLES

[0062] The subject matter of the present disclosure will be described in greater detail by way of specific examples. The following examples are provided to illustrate, but not to limit, the claimed subject matter. Example 1. Evaluation of AAV stuffers by FormBio

[0063] The example illustrates an evaluation of the adeno-associated virus (AAV) stuffer sequences by machine learning / artificial intelligence (ML / AI) based analysis FormBio.

[0064] Form Bio’s technology (https: / / www.formbio.com / ) comprises a complete and secure system for enabling computational biology at scale - from day-to-day bioinformatics work to artificial intelligence (AI) - accelerated therapeutic development.

[0065] A payload stuffer sequence (SEQ ID NO: 4) comprising a stuffer sequence from the 3’UTR of a human BMP-10 transcript (SEQ ID NO: 1; FIG.1), 4 payload sequences for gene augmentation therapy (SEQ ID Nos: 5-8), and 3 payload sequences for gene silencing therapy, were computationally evaluated by FormBio’s proprietary AI models for truncation propensity, deleterious 2° or 3° structures, and CpG island and GC content as a readout for immunotoxicity. As shown in FIGs.2-4, the designed payload sequences did not show any significant prediction for truncation propensity, deleterious secondary structures and CpG content (top panels) in comparison to a sub-optimal construct (bottom panels). These data demonstrates that the selected stuffer sequence is predicted to be a safe and inert sequence that can be used as a ‘stuffer’ in the context of AAV. The stuffer sequence is not predicted to have any negative impact on vector manufacturability and safety. Example 2. Validation of AAV stuffers in the lab

[0066] The example illustrates an evaluation of the adeno-associated virus (AAV) stuffer sequences by wet lab validation.

[0067] The designed payloads were synthesized on a plasmid vector backbone. Recombinant adeno-associated virus serotype 8 (rAAV8) and serotype 9 (rAAV9) were produced following standard triple transfection procedure using Fectovir (Sartorius, Cat# 101000004) in shake flask suspension culture. The cells and media used for packaging rAAV were Viral Production Cells 2.0 (ThermoFisher, Cat# A49784 and viral production media (Thermofisher, Cat# A4817901) respectively. After 72 hours of transfection, the produced viruses were harvested using Triton-X based lysis and subsequent affinity and ion exchange chromatography techniques were employed for purification.

[0068] Approximately 5E10 vg of each purified rAAV8 vector was run on an alkaline gel using standard protocol. As shown in FIGs. 5-6, single DNA bands at the expected size were observed in the alkaline gel. Four different payload sequences (SEQ ID Nos: 5-8) for gene augmentation therapy, indicated as ID# 1-4 in FIG.5C, each comprises a different combination of a promoter and codon optimized CDS sequences. The stuffer sequence in all four payloads is the same. Three different payload sequences for gene silencing therapy, indicated as ID# 1-3 in FIG. 6C, each is packaged with a different shRNA sequence in the payload. All other sequences in all three payloads for gene silencing therapy remain the same. A single band at the desired size, as shown in both FIGs. 5-6, indicates a homogenous product. It indicates that the stuffer sequence in a ssAAV8 vector did not introduce any truncated packaging that can lead to undesirable heterogenous product.

[0069] We further investigated stuffer sequences in a ssAAV9 vector for gene augmentation therapy and gene silencing therapy. Approximately 1E11 vg of each purified rAAV9 vector was run on an alkaline gel using standard protocol. As shown in FIG. 7A, single DNA bands at the expected size were observed in the alkaline gel. Two different payload sequences for gene augmentation therapy, indicated as ID# 1 and 2 in FIG. 7A, each comprises a different combination of a promoter and codon optimized CDS sequences, while the stuffer sequence in the two payloads is the same. Two different payload sequences for gene silencing therapy, indicated as ID# 1 and 2 in FIG.7B, each is packaged with a different shRNA sequence in the payload, while all other sequences in the payloads for gene silencing therapy remain the same. As shown in both FIGs. 7A and 7B, a single band of the desired size indicates a homogenous product. It indicates that the stuffer sequence in a ssAAV9 vector did not introduce any truncated packaging that can lead to undesirable heterogenous products.

[0070] These data show that the novel stuffer sequences enable efficient genome packaging in at least two AAV serotypes 8 and 9, as evidenced by the expected sizes in FIGs.5C, 6C, 7A, and 7B. The novel stuffer sequences are also expected to be effective in other AAV serotypes.

[0071] Furthermore, shake flask production of AAV8 and AAV9 yielded high titers (E13- E14 vg / L), indicating no negative impact of the stuffer sequences on manufacturability. In addition, AAV8 vectors exhibited a high proportion of full capsids (~80%), as measured by analytical ultracentrifugation and mass photometry. This further confirms the highly efficient manufacturability of AAVs containing the stuffer sequences. Example 3. Overview of manufacturing process of AAV employed at research scale

[0072] The example illustrates an exemplary AAV manufacturing process.

[0073] As illustrated in FIG. 8, AAV production in suspension shake flasks involves culturing suspension-adapted HEK293 cells in serum-free media under shaking conditions. Transient transfection is performed using three plasmids: the AAV transfer plasmid, Rep / Capplasmid, and adenoviral helper plasmid, at a cell density of 1–3×10 cells / m. The AAV transferplasmid contains the gene of interest flanked by AAV inverted terminal repeats (ITRs). The Rep / Cap plasmid supplies AAV Replication (Rep) and Capsid (Cap) proteins for the desired serotype. The adenoviral helper plasmid provides adenoviral genes (E2A, E4, and VA) necessary for AAV replication. Standard transfection reagents, such as polyethyleneimine (PEI) or similar compounds, are used in the transfection. After 48-72 h incubation, the cells are lysed with 0.5% triton-X in presence of Benzonase®. After clarification of the cell lysates by centrifugation, the supernatant is purified by standard affinity chromatography using AAVX resins, following which a polishing step is implemented via standard IEX chromatography using CIMmultus® QA Monolithic Column. The full AAV peak is collected and buffer exchanged with 1 x PBS + 0.1% Pluronic F68 and filter sterilized. This final product is further used for all downstream QC analysis and experiments. V. EXEMPLARY EMBODIMENTS

[0074] Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments:

[0075] Embodiment 1. A recombinant polynucleotide comprising a first nucleic acid sequence flanked by two inverted terminal repeat (ITR) sequences, wherein the first nucleic acid sequence encodes a 3' untranslated region (3'UTR) of a bone morphogenetic protein 10 (BMP-10) transcript.

[0076] Embodiment 2. The recombinant polynucleotide of embodiment 1, wherein the BMP-10 transcript is a human BMP-10 transcript.

[0077] Embodiment 3. The recombinant polynucleotide of embodiment 1 or 2, wherein the length of the recombinant polynucleotide is between about 3-5 kb, optionally between about 4-4.7 kb.

[0078] Embodiment 4. The recombinant polynucleotide of any one of embodiments 1- 3, wherein the first nucleic acid sequence comprises a sequence at least 80% identity to SEQ ID NO: 1.

[0079] Embodiment 5. The recombinant polynucleotide of any one of embodiments 1- 4, wherein the two ITR sequences are 5’ ITR comprising a sequence at least 80% identity to SEQ ID NO: 2 and 3’ ITR comprising a sequence at least 80% identity to SEQ ID NO: 3.

[0080] Embodiment 6. The recombinant polynucleotide of any one of embodiments 1- 5, comprising a nucleic acid sequence at least 80% identity to SEQ ID NO: 4.

[0081] Embodiment 7. The recombinant polynucleotide of any one of embodiments 1- 5, wherein at least a portion of the first nucleic acid sequence is replaced with a second nucleic acid sequence, and wherein the length of the recombinant polynucleotide keeps the same.

[0082] Embodiment 8. The recombinant polynucleotide of embodiment 7, wherein the second nucleic acid sequence encodes a protein of interest operably linked to a promoter.

[0083] Embodiment 9. A vector comprising the recombinant polynucleotide of any one of embodiments 1-8.

[0084] Embodiment 10. The vector of embodiment 9, wherein the vector is an adeno- associated virus (AAV) vector.

[0085] Embodiment 11. The vector of embodiment 10, wherein the AAV vector is an AAV serotype 8 (AAV8) vector or an AAV serotype 9 (AAV9) vector.

[0086] Embodiment 12. A pharmaceutical composition comprising a recombinant polynucleotide of any one of embodiments 1-8 or a vector of any one of embodiments 9-11, and a pharmaceutically acceptable carrier.

[0087] Embodiment 13. A method of preventing or treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 12.

[0088] Embodiment 14. A method of manufacturing the recombinant polynucleotide of any one of embodiments 1-8, comprising culturing a host cell comprising a rAAV vector, wherein the rAAV vector comprises the recombinant polynucleotide, under conditions suitable for rAAV production.

[0089] Embodiment 15. The method of embodiment 14, wherein the rAAV vector is a self-complementary AAV (scAAV) vector.

[0090] Embodiment 16. The method of embodiment 15, wherein the scAAV vector is a scAAV serotype 8 (scAAV8) vector or a scAAV serotype 9 (scAAV9) vector.

[0091] Embodiment 17. The method of embodiment 14, wherein the rAAV vector is a single-stranded AAV (ssAAV) vector.

[0092] Embodiment 18. The method of embodiment 17, wherein the ssAAV vector is a ssAAV serotype 8 (ssAAV8) vector or a ssAAV serotype 9 (ssAAV9) vector.

[0093] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. INFORMAL SEQUENCE LISTING SEQ ID NO: 1 Stuffer sequence from hBMP10-3’UTR GAAAACAACCCCTAGGCAAAATACAGTGTCAAAAGACACACACTTGTGTATGTA TATTTGTACATACATGAACTTACAAAAATCTTTTGGTTCTATGCAGGCAGGTTAT ACTCATCTACATGCATGACTCACTCAAATGTATGCATATTAAAGGCCAATGGTAT GTTCTCTGTTACCTCTTCTCTGAGTAGGATTTAAGTCAAGATAATTGACATCAGG GTTTTAATCATCCTAGGAAGTATTACCCTTCCTAAATACTCTGGAAATGTAAGTG TAGCTTTTGTCAGTTGAGTTTCTGTAGTTGCTCAGAGAGGAGGGGCCTGGCAAAG GATGCTTGAAGGGAAGGGTTAATTGATACAGAAACCAAATGGACTTAAGAGGTC TTAGAGGTACAACAGGAAAAAATGAACCCATGGAGGACCAATGTGCTCCAGAGG GCAACTCCTGGTCAAGGCAGGAAAACAGAAGAAACCAGCATGAAATCTGTTCCT GAGACAATAGGAAAGCAAGGCCATGAGTACTGGCCATTGCTGCTGCAGAGAGTA GAGGGGTTGGGAGGGTACTTGAAGGGGAGAAAGGGAGAAAGGGAAGAGGATAT TGATGTTATCCTCAGAGGCTATCCTCAGATGTGTCTGTAAAACCAAAAGTGGTTC TGTTTAAAGAATTAAATCTTTTGGACAAAATAGAAGTATCAAAGACTAAGAGGA GGAAAATCAGAAGGGGTCTGTCTGTTGGAAGAAAGGATGTTAATATCTTTCTTCA CCTGTTAACACTGTTCCCTTGTCTCAGCACCCCTGCTTTCCTCTGCTCCATCTTCTT CCTTCCCTCTTTCTTCTTGGCCCTAGTGCCTCTCACCTTCCTCCCATCCCTGTGTCC CTGCTCTTCCCTGGCCTTCATGATGCATGCTGTGAACTGGAAAGGGACACCTAAG TGCCTTTGCTCTCAGGGTGAAAGGAGTTAGTGTTCTAATTTCTAAATCTTCCCGTG ATATTTAATTTGATTTTATTTTAATAATATCCTTTATCAAATGTTTTGTTCATGAAG AATACTTAGATATAAGAGAAAAGAAAACCAGAATAAGTAAGTATAAGAAAAAG AAAACCAGAGTCCAAATGAATGGATTTAAATAAACGAATTTATGAATAGACACA CAAGCAAATGACTCAAAAGCCAAGCCTGAATTTTGAAATCATGATTGTTTCATGG TAGAAACCCAGTAACTTTCCCATCTTCTCATCTTACCATCTTCCCATTTTGCAACT GGCCAGGACTGAAAGTGGTGAGGAAGTGTTCTGCACAGGAAGGGTCCGGTGGGT TTTCCTGGCAGGTGGCAGCCTCCAGTCTTAACCCCTAACCATTTCCCTCTACCTGG CAAGCATATTCTGGGCTTTGTGCAGGAGGAACCAGAGCAGAGCAGAGCAGTTTC CAAACTGTTCCAGCATCTCCAGGCAGGGAACATGGATGGTTCCTGTTAGAACCAG GAATGTGGTTTCCAGTACGTCATCCCCCATTCACCCCTACTCATCTTAAGGAATG CTTCCTTAAGAAGTCTGACACTTTTTTCTTTTTTTTACCCTGGAAAACAATCAGTC CCCTTTTACAAATCTCTTAAGGTTATAAACTCAAACTGCACTAGACAATAGAGGC AAGGAAAACTATTTTGAAGAAGGCCCAATTTGAACCTCAGCTCGCTAGATCTGTT GTCTGCTGGGTGAATCCTGGGGAATTCATTGCTCATGGAAGAGTGGTGTTTTGAC CGTCAGTGGGTATACAGTGATTGATGCTTTGAGAAAGCCATGCAACTCAGAATCA CTATTTGTGGAACAGCCTCCATATAACATGATGTGTGTATGTGTGTGTGATGTAT AGCTGGCTTTGAATAACCACGCTTAATACCCTCAGTGGAACGTGGTGCATCCTTC ATACTCCATGGTACACAATGTATACTCACATACTCCAAGGTACTTCTTATCCTTTC TAAAGGACACTGTAATACATACACAGTAAAACCTCAGCTGACCAGGATAGTCCT GGTTAGATAAAAATTAAGCAAATTTTCTTTATGTCTAATACATATGACAGTGGTGTGCTGGTAAAACCCTTACAACCAGCAATCCAGGGGGAGAAAAAGCCCTGATTTG TGGATTTCAATGCTGCAAACGCTCCTACATGGTCTATCTTGAGCTGCCAAGGTGA TGTCAACTGGCTTGCAAAACTTCTAAAACCTAAGTCAGCTCTTGGGAGCTGTGCA CTGGTGCTGGCACACCGCTGACATTTAGTATCCCACTGACATTAGCACACCGCTG ACATTAGCACCCTGATACTAGAATGTTAAGGATGTGCCAGCCCTGCTTTCCACTG TGTGCGCTGCTGGCTCCGGGCTTCTGGAGAAATCTTGTCTCGAGCACTTATAGTC ACTCTTCTGTTAAAGAGATATGAAAATATAGGCTGTTGGCGATCAGGCTGCATAG CCCAGTTGTTTCTTTCTGCCCATCCAGATGTAAACCTGCATTTCTATGATTTTCTG CTTCCCTGTGGAAAGGGAGTGCAGAGAGCAACATATTGTTATTCAAATGTTGCTA GTTGTATTTTTTTGGCTTTTTGTCAGAAAGTGAAAAGACACAGCCCGTAGAGGAG TTCTCCCAGCTCCCACCGGAACCCCGGCAGCACACCGGTGTCACTTTCAACTCCT GCATTGACAGGAAGCCAGAATATTGTCAGATCCTTAAAAAGGTAGAATCTTACT AATGTTTCTCTGGTCATAGACCTTCTTCTGGGTCCCATGCCAGTTTTTTTCCCCCTT TTATTGCAGAATTATCATCACCGAAGTTGATATAGCTAAACAACTCTTGAGGAGA ATTATAGCAGAGGGACTTGGGCAGCCCACAGCCAAAATATTCATGTAAGAGTTT CTAGGCTTCCCACTATTTATTGATTGATATATTTATTTTTGTAAAATATTGTAGAA TACCAAATATTTTATTTTTATGTCTGTGATTCCCTTTTGGATAGAGAGTCTTCTGG GCTTCAGAGTATGGCTCACATATAATATATGGCCTGTATTTCCATCTCTCTCTTTT TGCATCAGAATATTCTATTGGTTTAAGGAATCTTGCCATAGCTTGCCATAATTTTG TATATTTGTGTCTTGCAGGCATGCTGTGCCTACAAGCATGCTTTGTAGTATATTTT CTGATTTGTAAATAGTACAGTTTTAACTGAAGTGTAGATGTTCCAGGAAATATTT AACCAATACATTGTCATGCAGTTTCATAAAGTGATGTTGTCAGTTTATCATGAGA AGCAAATATTGTCAGGCTGTGAGAGAATGTGATTGCTACTCAGTAAACGGAGAT GCTATTTAAATGAATCTTTTCATCCCCCAAATCTGGTACTTCCCCTCTGAATTCAC CTGTTGCCGTCACTGACCCAGAAATTCAGAGATGTGATTGGTCAAATTCATTTGT AATTGGACCAATTACTTTGTAAATGTCAAAAATGTTAGCTTAGTGATTTCCAAAA GAGTCAGCCCCAAAGTGCCGAATTCTTTCCTGATGTGTAAATTTTGGTCCGGGGA ATTAATGACAAAAACCTTTTTTTTTTAAGAACTGTTACTAAATGTTTCAGTATTTG TTATGATTTATATTCTTCAAGTAAAAGATATTTTCATATAAGAGAATTTTATGTAT TCTTCTTACTTGATTCATCAACAGATTATGAAGCCTAGCAGTGTTCTGGCAAAAC CTTTTTAACTTGCCTAGAACACAGAAGGGATGTTGCCCTTTTTAAGGTTGTAGAT CTTTTCAGAGTCACTGTGGGTCATTCTCAACCCTTTGCAGTTCTGCAAGCTCACAT ATGGTGCAAGAGGAAATGCTGGTATGACTCTGTGCATTTTCTTGCCCCCAAACAG GAACAGTTGATTTCCTACATTAAATGGAAGGTAGATGTGACTATGGAAGAGAAG TACACAGGGGTGTTTCTGCATGTGAAATATTTGTCATAAGTTAAAAACCCATCAT GGAATAACTGTTTGTTGTGATCAGCATGTTTTTATCACACATGGGCACTGGTTTTG TGAGCACCTGGCTTGATAGTGGTGGGGCGGCCAATGTGCCAGCGCCCGCCTACTG AGCATTCATGGACATTGGAAATCAGCGAACACCACAGACACACCGAATCAGCTG ATGCACGGTCATGTTCTGGGGTAAAACCTGTTCATTATCCATGTCCACATTTGTAT AGTTATCACTAGACTAAAATGAAGATGGAAATAAATATTTTACCCTGTGTTCATG AACCAAATGTGTACCTTCAGAGTCTTTTTAAGTAAAATAAAAAAA SEQ ID NO: 2 5’ AAV2 ITR cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtggccaactccatcactaggggttcct SEQ ID NO: 3 3’ AAV2 ITR Aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgc ccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggSEQ ID NO: 4 Payload Stuffer Sequence: 5’ ITR-Stuffer-3’ ITR cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagc gagcgcgcagagagggagtggccaactccatcactaggggttcctGAAAACAACCCCTAGGCAAAATACA GTGTCAAAAGACACACACTTGTGTATGTATATTTGTACATACATGAACTTACAAA AATCTTTTGGTTCTATGCAGGCAGGTTATACTCATCTACATGCATGACTCACTCAA ATGTATGCATATTAAAGGCCAATGGTATGTTCTCTGTTACCTCTTCTCTGAGTAGG ATTTAAGTCAAGATAATTGACATCAGGGTTTTAATCATCCTAGGAAGTATTACCC TTCCTAAATACTCTGGAAATGTAAGTGTAGCTTTTGTCAGTTGAGTTTCTGTAGTT GCTCAGAGAGGAGGGGCCTGGCAAAGGATGCTTGAAGGGAAGGGTTAATTGATA CAGAAACCAAATGGACTTAAGAGGTCTTAGAGGTACAACAGGAAAAAATGAACC CATGGAGGACCAATGTGCTCCAGAGGGCAACTCCTGGTCAAGGCAGGAAAACAG AAGAAACCAGCATGAAATCTGTTCCTGAGACAATAGGAAAGCAAGGCCATGAGT ACTGGCCATTGCTGCTGCAGAGAGTAGAGGGGTTGGGAGGGTACTTGAAGGGGA GAAAGGGAGAAAGGGAAGAGGATATTGATGTTATCCTCAGAGGCTATCCTCAGA TGTGTCTGTAAAACCAAAAGTGGTTCTGTTTAAAGAATTAAATCTTTTGGACAAA ATAGAAGTATCAAAGACTAAGAGGAGGAAAATCAGAAGGGGTCTGTCTGTTGGA AGAAAGGATGTTAATATCTTTCTTCACCTGTTAACACTGTTCCCTTGTCTCAGCAC CCCTGCTTTCCTCTGCTCCATCTTCTTCCTTCCCTCTTTCTTCTTGGCCCTAGTGCC TCTCACCTTCCTCCCATCCCTGTGTCCCTGCTCTTCCCTGGCCTTCATGATGCATG CTGTGAACTGGAAAGGGACACCTAAGTGCCTTTGCTCTCAGGGTGAAAGGAGTT AGTGTTCTAATTTCTAAATCTTCCCGTGATATTTAATTTGATTTTATTTTAATAATA TCCTTTATCAAATGTTTTGTTCATGAAGAATACTTAGATATAAGAGAAAAGAAAA CCAGAATAAGTAAGTATAAGAAAAAGAAAACCAGAGTCCAAATGAATGGATTTA AATAAACGAATTTATGAATAGACACACAAGCAAATGACTCAAAAGCCAAGCCTG AATTTTGAAATCATGATTGTTTCATGGTAGAAACCCAGTAACTTTCCCATCTTCTC ATCTTACCATCTTCCCATTTTGCAACTGGCCAGGACTGAAAGTGGTGAGGAAGTG TTCTGCACAGGAAGGGTCCGGTGGGTTTTCCTGGCAGGTGGCAGCCTCCAGTCTT AACCCCTAACCATTTCCCTCTACCTGGCAAGCATATTCTGGGCTTTGTGCAGGAG GAACCAGAGCAGAGCAGAGCAGTTTCCAAACTGTTCCAGCATCTCCAGGCAGGG AACATGGATGGTTCCTGTTAGAACCAGGAATGTGGTTTCCAGTACGTCATCCCCC ATTCACCCCTACTCATCTTAAGGAATGCTTCCTTAAGAAGTCTGACACTTTTTTCT TTTTTTTACCCTGGAAAACAATCAGTCCCCTTTTACAAATCTCTTAAGGTTATAAA CTCAAACTGCACTAGACAATAGAGGCAAGGAAAACTATTTTGAAGAAGGCCCAA TTTGAACCTCAGCTCGCTAGATCTGTTGTCTGCTGGGTGAATCCTGGGGAATTCA TTGCTCATGGAAGAGTGGTGTTTTGACCGTCAGTGGGTATACAGTGATTGATGCT TTGAGAAAGCCATGCAACTCAGAATCACTATTTGTGGAACAGCCTCCATATAACA TGATGTGTGTATGTGTGTGTGATGTATAGCTGGCTTTGAATAACCACGCTTAATA CCCTCAGTGGAACGTGGTGCATCCTTCATACTCCATGGTACACAATGTATACTCA CATACTCCAAGGTACTTCTTATCCTTTCTAAAGGACACTGTAATACATACACAGT AAAACCTCAGCTGACCAGGATAGTCCTGGTTAGATAAAAATTAAGCAAATTTTCT TTATGTCTAATACATATGACAGTGGTGTGCTGGTAAAACCCTTACAACCAGCAAT CCAGGGGGAGAAAAAGCCCTGATTTGTGGATTTCAATGCTGCAAACGCTCCTAC ATGGTCTATCTTGAGCTGCCAAGGTGATGTCAACTGGCTTGCAAAACTTCTAAAA CCTAAGTCAGCTCTTGGGAGCTGTGCACTGGTGCTGGCACACCGCTGACATTTAG TATCCCACTGACATTAGCACACCGCTGACATTAGCACCCTGATACTAGAATGTTA AGGATGTGCCAGCCCTGCTTTCCACTGTGTGCGCTGCTGGCTCCGGGCTTCTGGA GAAATCTTGTCTCGAGCACTTATAGTCACTCTTCTGTTAAAGAGATATGAAAATA TAGGCTGTTGGCGATCAGGCTGCATAGCCCAGTTGTTTCTTTCTGCCCATCCAGAT GTAAACCTGCATTTCTATGATTTTCTGCTTCCCTGTGGAAAGGGAGTGCAGAGAG CAACATATTGTTATTCAAATGTTGCTAGTTGTATTTTTTTGGCTTTTTGTCAGAAAGTGAAAAGACACAGCCCGTAGAGGAGTTCTCCCAGCTCCCACCGGAACCCCGGC AGCACACCGGTGTCACTTTCAACTCCTGCATTGACAGGAAGCCAGAATATTGTCA GATCCTTAAAAAGGTAGAATCTTACTAATGTTTCTCTGGTCATAGACCTTCTTCTG GGTCCCATGCCAGTTTTTTTCCCCCTTTTATTGCAGAATTATCATCACCGAAGTTG ATATAGCTAAACAACTCTTGAGGAGAATTATAGCAGAGGGACTTGGGCAGCCCA CAGCCAAAATATTCATGTAAGAGTTTCTAGGCTTCCCACTATTTATTGATTGATAT ATTTATTTTTGTAAAATATTGTAGAATACCAAATATTTTATTTTTATGTCTGTGAT TCCCTTTTGGATAGAGAGTCTTCTGGGCTTCAGAGTATGGCTCACATATAATATA TGGCCTGTATTTCCATCTCTCTCTTTTTGCATCAGAATATTCTATTGGTTTAAGGA ATCTTGCCATAGCTTGCCATAATTTTGTATATTTGTGTCTTGCAGGCATGCTGTGC CTACAAGCATGCTTTGTAGTATATTTTCTGATTTGTAAATAGTACAGTTTTAACTG AAGTGTAGATGTTCCAGGAAATATTTAACCAATACATTGTCATGCAGTTTCATAA AGTGATGTTGTCAGTTTATCATGAGAAGCAAATATTGTCAGGCTGTGAGAGAATG TGATTGCTACTCAGTAAACGGAGATGCTATTTAAATGAATCTTTTCATCCCCCAA ATCTGGTACTTCCCCTCTGAATTCACCTGTTGCCGTCACTGACCCAGAAATTCAG AGATGTGATTGGTCAAATTCATTTGTAATTGGACCAATTACTTTGTAAATGTCAA AAATGTTAGCTTAGTGATTTCCAAAAGAGTCAGCCCCAAAGTGCCGAATTCTTTC CTGATGTGTAAATTTTGGTCCGGGGAATTAATGACAAAAACCTTTTTTTTTTAAG AACTGTTACTAAATGTTTCAGTATTTGTTATGATTTATATTCTTCAAGTAAAAGAT ATTTTCATATAAGAGAATTTTATGTATTCTTCTTACTTGATTCATCAACAGATTAT GAAGCCTAGCAGTGTTCTGGCAAAACCTTTTTAACTTGCCTAGAACACAGAAGGG ATGTTGCCCTTTTTAAGGTTGTAGATCTTTTCAGAGTCACTGTGGGTCATTCTCAA CCCTTTGCAGTTCTGCAAGCTCACATATGGTGCAAGAGGAAATGCTGGTATGACT CTGTGCATTTTCTTGCCCCCAAACAGGAACAGTTGATTTCCTACATTAAATGGAA GGTAGATGTGACTATGGAAGAGAAGTACACAGGGGTGTTTCTGCATGTGAAATA TTTGTCATAAGTTAAAAACCCATCATGGAATAACTGTTTGTTGTGATCAGCATGT TTTTATCACACATGGGCACTGGTTTTGTGAGCACCTGGCTTGATAGTGGTGGGGC GGCCAATGTGCCAGCGCCCGCCTACTGAGCATTCATGGACATTGGAAATCAGCG AACACCACAGACACACCGAATCAGCTGATGCACGGTCATGTTCTGGGGTAAAAC CTGTTCATTATCCATGTCCACATTTGTATAGTTATCACTAGACTAAAATGAAGAT GGAAATAAATATTTTACCCTGTGTTCATGAACCAAATGTGTACCTTCAGAGTCTT TTTAAGTAAAATAAAAAAAaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactga ggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg SEQ ID NO: 5 Payload Sequence ID#1 for gene augmentation therapy 5’AAV2-ITR ‘Stuffer sequence’ from hBMP10-3’UTR MRE1 restriction site or cloning site hTNNT2 promoter SV40 intron Kozak sequence hATF4_native sequence SV40 polyA Not1 restriction site / cloning site 3’AAV2-ITR TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGG TCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCA GAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGAAAACAACCCCTAGGCAAAATACAGTGTCAAAAGACACACACTTGTGTATGTATATTTGTACATACATGAAC TTACAAAAATCTTTTGGTTCTATGCAGGCAGGTTATACTCATCTACATGCATGACT CACTCAAATGTATGCATATTAAAGGCCAATGGTATGTTCTCTGTTACCTCTTCTCT GAGTAGGATTTAAGTCAAGATAATTGACATCAGGGTTTTAATCATCCTAGGAAGT ATTACCCTTCCTAAATACTCTGGAAATGTAAGTGTAGCTTTTGTCAGTTGAGTTTC TGTAGTTGCTCAGAGAGGAGGGGCCTGGCAAAGGATGCTTGAAGGGAAGGGTTA ATTGATACAGAAACCAAATGGACTTAAGAGGTCTTAGAGGTACAACAGGAAAAA ATGAACCCATGGAGGACCAATGTGCTCCAGAGGGCAACTCCTGGTCAAGGCAGG AAAACAGAAGAAACCAGCATGAAATCTGTTCCTGAGACAATAGGAAAGCAAGG CCATGAGTACTGGCCATTGCTGCTGCAGAGAGTAGAGGGGTTGGGAGGGTACTT GAAGGGGAGAAAGGGAGAAAGGGAAGAGGATATTGATGTTATCCTCAGAGGCT ATCCTCAGATGTGTCTGTAAAACCAAAAGTGGTTCTGTTTAAAGAATTAAATCTT TTGGACAAAATAGAAGTATCAAAGACTAAGAGGAGGAAAATCAGAAGGGGTCT GTCTGTTGGAAGAAAGGATGTTAATATCTTTCTTCACCTGTTAACACTGTTCCCTT GTCTCAGCACCCCTGCTTTCCTCTGCTCCATCTTCTTCCTTCCCTCTTTCTTCTTGG CCCTAGTGCCTCTCACCTTCCTCCCATCCCTGTGTCCCTGCTCTTCCCTGGCCTTCA TGATGCATGCTGTGAACTGGAAAGGGACACCTAAGTGCCTTTGCTCTCAGGGTGA AAGGAGTTAGTGTTCTAATTTCTAAATCTTCCCGTGATATTTAATTTGATTTTATT TTAATAATATCCTTTATCAAATGTTTTGTTCATGAAGAATACTTAGATATAAGAG AAAAGAAAACCAGAATAAGTAAGTATAAGAAAAAGAAAACCAGAGTCCAAATG AATGGATTTAAATAAACGAATTTATGAATAGACACACAAGCAAATGACTCAAAA GCCAAGCCTGAATTTTGAAATCATGATTGTTTCATGGTAGAAACCCAGTAACTTT CCCATCTTCTCATCTTACCATCTTCCCATTTTGCAACTGGCCAGGACTGAAAGTGG TGAGGAAGTGTTCTGCACAGGAAGGGTCCGGTGGGTTTTCCTGGCAGGTGGCAG CCTCCAGTCTTAACCCCTAACCATTTCCCTCTACCTGGCAAGCATATTCTGGGCTT TGTGCAGGAGGAACCAGAGCAGAGCAGAGCAGTTTCCAAACTGTTCCAGCATCT CCAGGCAGGGAACATGGATGGTTCCTGTTAGAACCAGGAATGTGGTTTCCAGTA CGTCATCCCCCATTCACCCCTACTCATCTTAAGGACGCCGGCGAAGACCCCTAG GCCTGACCTATGCATCTGCAATCTAGTAGGTTTAGCAGATCATAGCCCCGCA CTGCTTGATTTTAAAGCCGTTAGGGGATGACCTTTGACAGTCCGCATCACCC CTCTCACACAACGAGCGCCTGTTCAAGGTTCTTGACTGGAAGTTCTACCTTG TATCTGGCCTCCTGTAGCAGTTTCAGTCCATTCCCTGTGAGGAGGGTGTGCC ACATGGCTTTGGGGGTCATGGAGAAGACCCACCTTGCAGATGTCCTCACTG GGGCTGGCAGAGCCGGCAACCTGCCTAAGGCTGCTCAGTCCATTAGGAGCC AGTAGCCTGGAAGATGTCTTTACCCCCAGCATCAGTTCAAGTGGAGCAGCA CATAACTCTTGCCCTCTGCCTTCCAAGATTCTGGTGCTGAGACTTATGGAGT GTCTTGGAGGTTGCCTTCTGCCCCCCAACCCTGCTCCCAGCTGGCCCTCCCA GGCCTGGGTTGCTGGCCTCTGCTTTATCAGGATTCTCAAGAGGGACAGCTG GTTTATGTTGCATGACTGTTCCCTGCATATCTGCTCTGGTTTTAAATAGCTTA TCTGAGCAGCTGGAGGACCACATGGGCTTATATGGCGTGGGGTACATGTTC CTGTAGCCTTGTCCCTGGCACCTGCCAAAATAGCAGCCAACACCCCCCACCC CCACCGCCATCCCCCTGCCCCACCCGTCCCCTGTCGCACATTCCTCCCTCCG CAGGGCTGGCTCACCAGGCCCCAGCCCACATGCCTGCTTAAAGCCCTCTCC ATCCTCTGCCTCACCCAGTCCCCGCTGAGACTGAGCAGACGCCTCCAGGAT CTGTCGGCAGCTGCTGTTCTGAGGTAAGGCTCGGGCAGGGCTCTGGGGAAG AGGAGAGCAGAGAATGGACGGGGAGATGTGAGGGTCTTGGGCCCTGGCAT IJĵATTTACCCAGAGTCTGCCTGTGTCCGCAGAAGTCCATGGCCCCTCCTGGTGG AGGCCACACTTCAGAGGACAGGTTGCCAGGTCTGGGCTCCAAGATTGGTAC AATAGAGCAGAGAGAAAGAGAGAATCCCATGGTAGCCATTGGAGACTAGGG TTGGAGTCCTGGACCTGCCACTAAAGATGTAGAGTTTAAACTCTTGGATGAG TCACTTTCCCTCCCCAGGCCTCAGTTTTCTCATCTGTAGAGTGGGCTAAATA TTTGCTAGGCCTCTGCCTGCTCTAAATACAATGAGGCTCTAACCAACAGAGACTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCG GCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA SEQ ID NO: 6 Payload Sequence ID#2 for gene augmentation therapy 5’AAV2-ITR ‘Stuffer sequence’ from hBMP10-3’UTR MRE1 restriction site or cloning site hMybpc3 promoter SV40 intron Kozak sequencesequenceSV40 polyA Not1 restriction site / cloning site 3’AAV2-ITR TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGG TCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCA GAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTACAGTGTCAAAAGACACA CACTTGTGTATGTATATTTGTACATACATGAACTTACAAAAATCTTTTGGTTCTAT GCAGGCAGGTTATACTCATCTACATGCATGACTCACTCAAATGTATGCATATTAA AGGCCAATGGTATGTTCTCTGTTACCTCTTCTCTGAGTAGGATTTAAGTCAAGAT AATTGACATCAGGGTTTTAATCATCCTAGGAAGTATTACCCTTCCTAAATACTCT GGAAATGTAAGTGTAGCTTTTGTCAGTTGAGTTTCTGTAGTTGCTCAGAGAGGAG GGGCCTGGCAAAGGATGCTTGAAGGGAAGGGTTAATTGATACAGAAACCAAATG GACTTAAGAGGTCTTAGAGGTACAACAGGAAAAAATGAACCCATGGAGGACCAA TGTGCTCCAGAGGGCAACTCCTGGTCAAGGCAGGAAAACAGAAGAAACCAGCAT GAAATCTGTTCCTGAGACAATAGGAAAGCAAGGCCATGAGTACTGGCCATTGCT GCTGCAGAGAGTAGAGGGGTTGGGAGGGTACTTGAAGGGGAGAAAGGGAGAAA GGGAAGAGGATATTGATGTTATCCTCAGAGGCTATCCTCAGATGTGTCTGTAAAA CCAAAAGTGGTTCTGTTTAAAGAATTAAATCTTTTGGACAAAATAGAAGTATCAA AGACTAAGAGGAGGAAAATCAGAAGGGGTCTGTCTGTTGGAAGAAAGGATGTTA ATATCTTTCTTCACCTGTTAACACTGTTCCCTTGTCTCAGCACCCCTGCTTTCCTCT GCTCCATCTTCTTCCTTCCCTCTTTCTTCTTGGCCCTAGTGCCTCTCACCTTCCTCC CATCCCTGTGTCCCTGCTCTTCCCTGGCCTTCATGATGCATGCTGTGAACTGGAAA GGGACACCTAAGTGCCTTTGCTCTCAGGGTGAAAGGAGTTAGTGTTCTAATTTCT AAATCTTCCCGTGATATTTAATTTGATTTTATTTTAATAATATCCTTTATCAAATG TTTTGTTCATGAAGAATACTTAGATATAAGAGAAAAGAAAACCAGAATAAGTAA GTATAAGAAAAAGAAAACCAGAGTCCAAATGAATGGATTTAAATAAACGAATTT ATGAATAGACACACAAGCAAATGACTCAAAAGCCAAGCCTGAATTTTGAAATCA TGATTGTTTCATGGTAGAAACCCAGTAACTTTCCCATCTTCTCATCTTACCATCTT CCCATTTTGCAACTGGCCAGGACTGAAAGTGGTGAGGAAGTGTTCTGCACAGGA AGGGTCCGGTGGGTTTTCCTGGCAGGTGGCAGCCTCCAGTCTTAACCCCTAACCA TTTCCCTCTACCTGGCAAGCATATTCTGGGCTTTGTGCAGGAGGAACCAGAGCAG AGCAGAGCAGTTTCCAAACTGTTCCAGCATCTCCAGGCAGGGAACATGGATGGT TCCTGTTAGAACCAGGAATGTGGTTTCCAGTACGTCATCCCCCATTCACCCCTACT CATCTTAAGGAATGCTTCCTTAAGAAGTCTGACACTTTTTTCTTTTTTTTACCCTG GAAAACAATCAGTCCCCTTTTACAAATCTCTTAAGGTTATAAACTCAAACTGCAC TAGACAATAGAGGCAAGGAAAACTATTTTGAAGAAGGCCCAATTTGAACCTCAG CTCGCTAGATCTGTTGTCTGCTGGGTGAATCCTGGGGAATTCATTGCTCATGGAA GAGTGGTGTTTTGACCGTCAGTGGGTATACAGTGATTGATGCTTTGAGAAAGCCA TGCAACTCAGAATCACTATTTGTGGAACAGCCTCCGCCGGCGCATACGTGTGAC AATCCTGTGCAGTGGGTGCTGCTTTCACCCACCCCGCTTTACAGATGAGGAA ACCAGGTGCTTTGAGATTAAAGAGATGGCAGTGCTAGGACTCAGAGACACC CTGGCCCCAGGGTCTGTGGACATGACCACTGCCTGAACTGCCTGCGAGGAT GCTCCTGACCCGTTAGCCTTAGGGTTATGGGCCCAGCATGCTCCCACCGTAC AATTTGCTGTAAGAGGGACACACACGGGGCTTGGAGTACCCCTGAGGCTTG CCAAAGGTTGGGGAGGCAGAATTGTGCTGCGGGGGGTGAGGGGGGAAGGGACAGGAGCCAGCCAGGGACAATGTGGCCCTACCTTCTCCCTGGAGAGACCT CAGCTCTCTGGAATTCATCTATATTTAGCAGGTGGCTGGACAGGAGGCAGAT AAGCAGAGCCTGGGGAGGGGGGAGGTCCCCATATATAGTGGGAAGGACAG GACCCCACTCAGTCCCTCTTTGGGTGACCTGTGCCTGCTTCGTGCCTGGTGT GACGTCTCTCAGGATGCCTGAGCCGGGGAAGAAGCCAGGTAGCTTTAGGAC TGGGGTTGGGTCTAAGTGTGGGAGCAGGGGGGTGTCTACAATTGGGGAGCA GGGCTAGGAGGGAGTTCTTGAGGGGGTTGACGAGGACGTGGCCTCTGAGCC CCAGGACAGGGGCAGCCAGTCCTCCAGGGTCCTTTTTTGATTCTCGGTTTCT TTTTCCCATCCCTCTGATGAGAGGTGAAGGTAAACCCAAGGTCAGGGACGC AGAGAGGGGACACAGCCAAGACAGGAAGCACCCGGGGTCAACCCATTCTGA GAATGCAAAGGGACCCCTGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGA TCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGCAAACCTCTACAAATGTGGTATGGCTGATTAGCGGCCGCAGGAACCCCTAGTGATGGA GTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAG CCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAA SEQ ID NO: 7 Payload Sequence ID#3 for gene augmentation therapy 5’AAV2-ITR ‘Stuffer sequence’ from hBMP10-3’UTR MRE1 restriction site or cloning site hMybpc3 promoterSV40 intron Kozak sequence hATF4_codon optimized sequence-1 SV40 polyA Not1 restriction site / cloning site 3’AAV2-ITR TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGG TCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCA GAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTACAGTGTCAAAAGACACA CACTTGTGTATGTATATTTGTACATACATGAACTTACAAAAATCTTTTGGTTCTAT GCAGGCAGGTTATACTCATCTACATGCATGACTCACTCAAATGTATGCATATTAA AGGCCAATGGTATGTTCTCTGTTACCTCTTCTCTGAGTAGGATTTAAGTCAAGAT AATTGACATCAGGGTTTTAATCATCCTAGGAAGTATTACCCTTCCTAAATACTCT GGAAATGTAAGTGTAGCTTTTGTCAGTTGAGTTTCTGTAGTTGCTCAGAGAGGAG GGGCCTGGCAAAGGATGCTTGAAGGGAAGGGTTAATTGATACAGAAACCAAATG GACTTAAGAGGTCTTAGAGGTACAACAGGAAAAAATGAACCCATGGAGGACCAA TGTGCTCCAGAGGGCAACTCCTGGTCAAGGCAGGAAAACAGAAGAAACCAGCAT GAAATCTGTTCCTGAGACAATAGGAAAGCAAGGCCATGAGTACTGGCCATTGCT GCTGCAGAGAGTAGAGGGGTTGGGAGGGTACTTGAAGGGGAGAAAGGGAGAAA GGGAAGAGGATATTGATGTTATCCTCAGAGGCTATCCTCAGATGTGTCTGTAAAA CCAAAAGTGGTTCTGTTTAAAGAATTAAATCTTTTGGACAAAATAGAAGTATCAA AGACTAAGAGGAGGAAAATCAGAAGGGGTCTGTCTGTTGGAAGAAAGGATGTTA ATATCTTTCTTCACCTGTTAACACTGTTCCCTTGTCTCAGCACCCCTGCTTTCCTCT GCTCCATCTTCTTCCTTCCCTCTTTCTTCTTGGCCCTAGTGCCTCTCACCTTCCTCC CATCCCTGTGTCCCTGCTCTTCCCTGGCCTTCATGATGCATGCTGTGAACTGGAAA GGGACACCTAAGTGCCTTTGCTCTCAGGGTGAAAGGAGTTAGTGTTCTAATTTCT AAATCTTCCCGTGATATTTAATTTGATTTTATTTTAATAATATCCTTTATCAAATG TTTTGTTCATGAAGAATACTTAGATATAAGAGAAAAGAAAACCAGAATAAGTAA GTATAAGAAAAAGAAAACCAGAGTCCAAATGAATGGATTTAAATAAACGAATTT ATGAATAGACACACAAGCAAATGACTCAAAAGCCAAGCCTGAATTTTGAAATCA TGATTGTTTCATGGTAGAAACCCAGTAACTTTCCCATCTTCTCATCTTACCATCTT CCCATTTTGCAACTGGCCAGGACTGAAAGTGGTGAGGAAGTGTTCTGCACAGGA AGGGTCCGGTGGGTTTTCCTGGCAGGTGGCAGCCTCCAGTCTTAACCCCTAACCA TTTCCCTCTACCTGGCAAGCATATTCTGGGCTTTGTGCAGGAGGAACCAGAGCAG AGCAGAGCAGTTTCCAAACTGTTCCAGCATCTCCAGGCAGGGAACATGGATGGT TCCTGTTAGAACCAGGAATGTGGTTTCCAGTACGTCATCCCCCATTCACCCCTACT CATCTTAAGGAATGCTTCCTTAAGAAGTCTGACACTTTTTTCTTTTTTTTACCCTG GAAAACAATCAGTCCCCTTTTACAAATCTCTTAAGGTTATAAACTCAAACTGCAC TAGACAATAGAGGCAAGGAAAACTATTTTGAAGAAGGCCCAATTTGAACCTCAG CTCGCTAGATCTGTTGTCTGCTGGGTGAATCCTGGGGAATTCATTGCTCATGGAA GAGTGGTGTTTTGACCGTCAGTGGGTATACAGTGATTGATGCTTTGAGAAAGCCA TGCAACTCAGAATCACTATTTGTGGAACAGCCTCCGCCGGCGCATACGTGTGAC AATCCTGTGCAGTGGGTGCTGCTTTCACCCACCCCGCTTTACAGATGAGGAA ACCAGGTGCTTTGAGATTAAAGAGATGGCAGTGCTAGGACTCAGAGACACC CTGGCCCCAGGGTCTGTGGACATGACCACTGCCTGAACTGCCTGCGAGGATGCTCCTGACCCGTTAGCCTTAGGGTTATGGGCCCAGCATGCTCCCACCGTAC AATTTGCTGTAAGAGGGACACACACGGGGCTTGGAGTACCCCTGAGGCTTG CCAAAGGTTGGGGAGGCAGAATTGTGCTGCGGGGGGTGAGGGGGGAAGGG ACAGGAGCCAGCCAGGGACAATGTGGCCCTACCTTCTCCCTGGAGAGACCT CAGCTCTCTGGAATTCATCTATATTTAGCAGGTGGCTGGACAGGAGGCAGAT AAGCAGAGCCTGGGGAGGGGGGAGGTCCCCATATATAGTGGGAAGGACAG GACCCCACTCAGTCCCTCTTTGGGTGACCTGTGCCTGCTTCGTGCCTGGTGT GACGTCTCTCAGGATGCCTGAGCCGGGGAAGAAGCCAGGTAGCTTTAGGAC TGGGGTTGGGTCTAAGTGTGGGAGCAGGGGGGTGTCTACAATTGGGGAGCA GGGCTAGGAGGGAGTTCTTGAGGGGGTTGACGAGGACGTGGCCTCTGAGCC CCAGGACAGGGGCAGCCAGTCCTCCAGGGTCCTTTTTTGATTCTCGGTTTCT TTTTCCCATCCCTCTGATGAGAGGTGAAGGTAAACCCAAGGTCAGGGACGC AGAGAGGGGACACAGCCAAGACAGGAAGCACCCGGGGTCAACCCATTCTGA GAATGCAAAGGGACCCCTGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGA TCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGA GAGGGAGTGGCCAA SEQ ID NO: 8 Payload Sequence ID#4 for gene augmentation therapy 5’AAV2-ITR‘Stuffer sequence’ from hBMP10-3’UTR MRE1 restriction site or cloning site hMybpc3 promoter SV40 intron Kozak sequence hATF4_codon optimized sequence-2 SV40 polyA Not1 restriction site / cloning site 3’AAV2-ITR TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGG TCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCA GAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTACAGTGTCAAAAGACACA CACTTGTGTATGTATATTTGTACATACATGAACTTACAAAAATCTTTTGGTTCTAT GCAGGCAGGTTATACTCATCTACATGCATGACTCACTCAAATGTATGCATATTAA AGGCCAATGGTATGTTCTCTGTTACCTCTTCTCTGAGTAGGATTTAAGTCAAGAT AATTGACATCAGGGTTTTAATCATCCTAGGAAGTATTACCCTTCCTAAATACTCT GGAAATGTAAGTGTAGCTTTTGTCAGTTGAGTTTCTGTAGTTGCTCAGAGAGGAG GGGCCTGGCAAAGGATGCTTGAAGGGAAGGGTTAATTGATACAGAAACCAAATG GACTTAAGAGGTCTTAGAGGTACAACAGGAAAAAATGAACCCATGGAGGACCAA TGTGCTCCAGAGGGCAACTCCTGGTCAAGGCAGGAAAACAGAAGAAACCAGCAT GAAATCTGTTCCTGAGACAATAGGAAAGCAAGGCCATGAGTACTGGCCATTGCT GCTGCAGAGAGTAGAGGGGTTGGGAGGGTACTTGAAGGGGAGAAAGGGAGAAA GGGAAGAGGATATTGATGTTATCCTCAGAGGCTATCCTCAGATGTGTCTGTAAAA CCAAAAGTGGTTCTGTTTAAAGAATTAAATCTTTTGGACAAAATAGAAGTATCAA AGACTAAGAGGAGGAAAATCAGAAGGGGTCTGTCTGTTGGAAGAAAGGATGTTA ATATCTTTCTTCACCTGTTAACACTGTTCCCTTGTCTCAGCACCCCTGCTTTCCTCT GCTCCATCTTCTTCCTTCCCTCTTTCTTCTTGGCCCTAGTGCCTCTCACCTTCCTCC CATCCCTGTGTCCCTGCTCTTCCCTGGCCTTCATGATGCATGCTGTGAACTGGAAA GGGACACCTAAGTGCCTTTGCTCTCAGGGTGAAAGGAGTTAGTGTTCTAATTTCT AAATCTTCCCGTGATATTTAATTTGATTTTATTTTAATAATATCCTTTATCAAATG TTTTGTTCATGAAGAATACTTAGATATAAGAGAAAAGAAAACCAGAATAAGTAA GTATAAGAAAAAGAAAACCAGAGTCCAAATGAATGGATTTAAATAAACGAATTT ATGAATAGACACACAAGCAAATGACTCAAAAGCCAAGCCTGAATTTTGAAATCA TGATTGTTTCATGGTAGAAACCCAGTAACTTTCCCATCTTCTCATCTTACCATCTT CCCATTTTGCAACTGGCCAGGACTGAAAGTGGTGAGGAAGTGTTCTGCACAGGA AGGGTCCGGTGGGTTTTCCTGGCAGGTGGCAGCCTCCAGTCTTAACCCCTAACCA TTTCCCTCTACCTGGCAAGCATATTCTGGGCTTTGTGCAGGAGGAACCAGAGCAG AGCAGAGCAGTTTCCAAACTGTTCCAGCATCTCCAGGCAGGGAACATGGATGGT TCCTGTTAGAACCAGGAATGTGGTTTCCAGTACGTCATCCCCCATTCACCCCTACT CATCTTAAGGAATGCTTCCTTAAGAAGTCTGACACTTTTTTCTTTTTTTTACCCTG GAAAACAATCAGTCCCCTTTTACAAATCTCTTAAGGTTATAAACTCAAACTGCAC TAGACAATAGAGGCAAGGAAAACTATTTTGAAGAAGGCCCAATTTGAACCTCAG CTCGCTAGATCTGTTGTCTGCTGGGTGAATCCTGGGGAATTCATTGCTCATGGAA GAGTGGTGTTTTGACCGTCAGTGGGTATACAGTGATTGATGCTTTGAGAAAGCCA TGCAACTCAGAATCACTATTTGTGGAACAGCCTCCGCCGGCGCATACGTGTGACAATCCTGTGCAGTGGGTGCTGCTTTCACCCACCCCGCTTTACAGATGAGGAA ACCAGGTGCTTTGAGATTAAAGAGATGGCAGTGCTAGGACTCAGAGACACC CTGGCCCCAGGGTCTGTGGACATGACCACTGCCTGAACTGCCTGCGAGGAT GCTCCTGACCCGTTAGCCTTAGGGTTATGGGCCCAGCATGCTCCCACCGTAC AATTTGCTGTAAGAGGGACACACACGGGGCTTGGAGTACCCCTGAGGCTTG CCAAAGGTTGGGGAGGCAGAATTGTGCTGCGGGGGGTGAGGGGGGAAGGG ACAGGAGCCAGCCAGGGACAATGTGGCCCTACCTTCTCCCTGGAGAGACCT CAGCTCTCTGGAATTCATCTATATTTAGCAGGTGGCTGGACAGGAGGCAGAT AAGCAGAGCCTGGGGAGGGGGGAGGTCCCCATATATAGTGGGAAGGACAG GACCCCACTCAGTCCCTCTTTGGGTGACCTGTGCCTGCTTCGTGCCTGGTGT GACGTCTCTCAGGATGCCTGAGCCGGGGAAGAAGCCAGGTAGCTTTAGGAC TGGGGTTGGGTCTAAGTGTGGGAGCAGGGGGGTGTCTACAATTGGGGAGCA GGGCTAGGAGGGAGTTCTTGAGGGGGTTGACGAGGACGTGGCCTCTGAGCC CCAGGACAGGGGCAGCCAGTCCTCCAGGGTCCTTTTTTGATTCTCGGTTTCT TTTTCCCATCCCTCTGATGAGAGGTGAAGGTAAACCCAAGGTCAGGGACGC AGAGAGGGGACACAGCCAAGACAGGAAGCACCCGGGGTCAACCCATTCTGA GAATGCAAAGGGACCCCTGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGA TCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGCGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGG GCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGA GCGCGCAGAGAGGGAGTGGCCAA

Claims

WHAT IS CLAIMED IS:

1. A recombinant polynucleotide comprising a first nucleic acid sequence flanked by two inverted terminal repeat (ITR) sequences, wherein the first nucleic acid sequence encodes a 3' untranslated region (3'UTR) of a bone morphogenetic protein 10 (BMP-10) transcript.

2. The recombinant polynucleotide of claim 1, wherein the BMP-10 transcript is a human BMP-10 transcript.

3. The recombinant polynucleotide of claim 1, wherein the length of the recombinant polynucleotide is between about 3-5 kb, optionally between about 4-4.7 kb.

4. The recombinant polynucleotide of claim 1, wherein the first nucleic acid sequence comprises a sequence at least 80% identity to SEQ ID NO:

1.

5. The recombinant polynucleotide of claim 1, wherein the two ITR sequences are 5’ ITR comprising a sequence at least 80% identity to SEQ ID NO: 2 and 3’ ITR comprising a sequence at least 80% identity to SEQ ID NO:

3.

6. The recombinant polynucleotide of claim 1, comprising a nucleic acid sequence at least 80% identity to SEQ ID NO:

4. The recombinant polynucleotide of claim 1, wherein at least a portion of the first nucleic acid sequence is replaced with a second nucleic acid sequence, and wherein the length of the recombinant polynucleotide keeps the same.

8. The recombinant polynucleotide of claim 7, wherein the second nucleic acid sequence encodes a protein of interest operably linked to a promoter.

9. A vector comprising the recombinant polynucleotide of claim 1.

10. The vector of claim 9, wherein the vector is an adeno-associated virus (AAV) vector.

11. The vector of claim 10, wherein the AAV vector is an AAV serotype 8 (AAV8) vector or an AAV serotype 9 (AAV9) vector.

12. A pharmaceutical composition comprising a recombinant polynucleotide of claim 1 or a vector of claim 9, and a pharmaceutically acceptable carrier.

13. A method of preventing or treating a disease in a subject, comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 12.

14. A method of manufacturing the recombinant polynucleotide of claim 1, comprising: culturing a host cell comprising a rAAV vector, wherein the rAAV vector comprises the recombinant polynucleotide, under conditions suitable for rAAV production.

15. The method of claim 14, wherein the rAAV vector is a self-complementary AAV (scAAV) vector.

16. The method of claim 15, wherein the scAAV vector is a scAAV serotype 8 (scAAV8) vector or a scAAV serotype 9 (scAAV9) vector.

17. The method of claim 14, wherein the rAAV vector is a single-stranded AAV (ssAAV) vector.

18. The method of claim 17, wherein the ssAAV vector is a ssAAV serotype 8 (ssAAV8) vector or a ssAAV serotype 9 (ssAAV9) vector.

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