Cardioprotective compositions and methods for atrial fibrillation

Abstract

The present technology provides compositions and methods for gene therapy and / or cell therapy for treatment of heart diseases, e.g., atrial fibrillation. In particular, the present technology provides transgenes, expression cassettes, vectors, recombinant AAV (rAAV) viral genomes, rAAV viruses or virions, and pharmaceutical compositions for overexpressing synaptopodin 2 like (SYNPO2L), isoform A (SYNPO2LA) in a cardiac cell in a subject, as well as methods of using the same in the prevention or treatment atrial fibrillation.

Description

Attorney Docket No. TENA-054 / 01WO 334682-2486 CARDIOPROTECTIVE COMPOSITIONS AND METHODS FOR ATRIAL FIBRILLATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 622,217 filed January 18, 2024, the entire disclosure of which is incorporated herein in its entirety. REFERENCE TO THE ELECTRONIC SEQUENCE LISTING

[0002] The present application is being filed electronically via USPTO Patent Center and includes an electronically submitted Sequence Listing in XML format. The name of the XML file containing the Sequence Listing is TENA_054_01WO_SeqList_ST26.xml. The XML file, created on January 17, 2025, is 248,806 bytes in size and is being submitted electronically via USPTO Patent Center. The contents of the electronic Sequence Listing are incorporated by reference herein in their entirety. TECHNICAL FIELD

[0003] The present technology relates to cardioprotective compositions and methods for preventing and / or treating heart diseases (e.g., atrial fibrillation), for example, through increasing levels of the synaptopodin 2 like (SYNPO2L) gene, isoform A, in cardiac cells. BACKGROUND

[0004] The SYNPO2L gene encodes an actin-associated protein that plays a role in modulating actin-cytoskeleton mediated cell shape and conformation. SYNPO2L is expressed predominantly in the heart (mainly in cardiomyocytes) and has two different isoforms, isoform A and isoform B. SYNPO2L isoform B (also referred to as SYNPO2LB), which is primarily expressed in embryonic cardiac tissue, is a shorter isoform with 2 exons and contains a nuclear localization signal (NLS). SYNPO2L isoform A (also referred to as SYNPO2LA), which is primarily expressed in adult cardiac tissue, is a longer isoform with 4 exons and contains an NLS and a PDZ domain. SYNPO2L is localized to the Z-disk and is known to bind α-actinin. Mutations in the SYNPO2L gene have been associated with heart diseases including dilated cardiomyopathy and familialAttorney Docket No. TENA-054 / 01WO 334682-2486 atrial fibrillation. For example, overexpression of the short / fetal isoform (SYNPO2LB) can lead to contractile dysfunction and sarcomere disarray. Therefore, gene therapies and / or cell therapies targeting the SYNPO2L gene for treatment of heart diseases are needed. SUMMARY

[0005] The present technology provides compositions and methods for the prevention and / or treatment of heart diseases, e.g., atrial fibrillation. In particular, the present technology provides transgenes, expression cassettes, vectors, recombinant AAV (rAAV) viral genomes, rAAV viruses or virions, and pharmaceutical compositions for overexpressing SYNPO2LA in a cardiac cell in a subject, as well as methods of using the same in the prevention or treatment atrial fibrillation.

[0006] In some aspects, provided is a method of preventing and / or treating atrial fibrillation in a subject in need thereof, the method comprising overexpressing SYNPO2LA in a cardiac cell in the subject, for example, by administering to the subject a therapeutically effective amount of (i) a vector comprising a polynucleotide encoding a SYNPO2LA polypeptide operatively linked to a cardiac-specific promoter, (ii) a recombinant virion comprising the vector, or (iii) or a cell comprising the vector.

[0007] In some embodiments, the subject has had a prior heart disease selected from ischemic heart disease, heart failure, coronary artery disease, cardiomyopathy, pericarditis, and congenital heart disease.

[0008] In some embodiments, the subject has a genetic variant in the SYNPO2L gene that leads to increased expression of SYNPO2LB and / or decreased expression of SYNPO2LA. In some embodiments, the genetic variant in the SYNPO2L gene comprises a single-nucleotide polymorphism (SNP), for example, rs766868752.

[0009] In some embodiments, the SYNPO2LA polypeptide comprises an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2, and / or the polynucleotide encoding the SYNPO2LA polypeptide comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0010] In some embodiments, the cardiac-specific promoter is a troponin T (TNNT2) promoter or a chimeric variant thereof. In some embodiments, the TNNT2 promoter or chimeric variant thereof comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 5-16.

[0011] In some embodiments, the vector is an adeno-associated virus (AAV) vector; and / or the recombinant virion is a recombinant AAV (rAAV) virion. In some embodiments, the AAV vector is an AAV9 vector; and / or the rAAV virion comprises an AAV9 capsid protein or a variant thereof described herein.

[0012] In some embodiments, the method results in: (a) increased expression of SYNPO2LA in the cardiac cell in the subject, optionally wherein the cardiac cell is a cardiomyocyte; (b) decreased expression of SYNPO2LB in the cardiac cell in the subject, optionally wherein the cardiac cell is a cardiomyocyte; (c) increased atrial contractility; (d) increased cardiac conduction; (e) improvement in one or more symptoms associated with atrial fibrillation selected from irregular heartbeat or arrhythmia, heart palpitation, angina or chest pain, dizziness or lightheadedness, swelling, fainting, fatigue or weakness, and shortness of breath; and / or (f) reduced risk for blood clot, stroke, heart failure, and / or dementia.

[0013] In some embodiments, the administering is systemic administration or local administration to the heart. In some embodiments, the systemic administration is intravenous administration. In some embodiments, the local administration is by direct injection into the heart or cardiac tissue, intracoronary administration, or retrograde coronary sinus infusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG.1 shows the location of the rs766868752 splice variant mutation on the genome and its relation to the two isoforms of SYNPO2L.

[0015] FIGS. 2A-2B show significantly reduced SYNPO2LA mRNA and protein levels in an induced pluripotent stem cell (iPSC)-derived ventricular cell line (FIG.2A) and an iPSC-derived atrial cell line (FIG.2B) that are homozygous for the rs766868752 mutation, compared to control wild-type isogenic cells (WTC). FIG.2A further shows that SYNPO2LB protein levels are increased in the ventricular cell line. In the graphs, barsAttorney Docket No. TENA-054 / 01WO 334682-2486 appear in the following order from left to right: WTC, SYNPO2L Mut+ / +, WTC, SYNPO2L Mut+ / +.

[0016] FIGS.3A-3H show electrophysiological function in SYNPO2L Mut+ / +iPSC- derived atrial and ventricular cardiomyocytes compared to WTC. FIG. 3A shows representative action potential traces, FIG. 3B shows APDC90, FIG. 3C shows field potential spike amplitudes, and FIG.3D shows field potential spike slopes, for iPSC- derived atrial cardiomyocytes. FIG. 3E shows APDC90, FIG. 3F shows field potential spike amplitudes, and FIG. 3G shows field potential spike slopes, for iPSC-derived ventricular cardiomyocytes. FIG.3H shows beat period variation in SYNPO2L Mut+ / +and WTC iPSC-derived atrial cardiomyocytes.

[0017] FIGS.4A-4H show electrophysiological function in SYNPO2L Mut+ / +iPSC- derived atrial and ventricular cardiomyocytes compared to WTC. FIG.4A shows trace of calcium transients, FIG.4B shows calcium transient amplitudes, FIG.4C shows calcium influx rate, and FIG. 4D shows calcium reuptake rate, for iPSC-derived atrial cardiomyocytes. FIG.4E shows calcium transient amplitudes, FIG.4F shows calcium influx rate, and FIG. 4G shows calcium reuptake rate, for iPSC-derived ventricular cardiomyocytes. FIG.4H shows RNA-seq analysis of ion channel and conduction genes.

[0018] FIGS.5A-5J show contractility in SYNPO2L Mut+ / +engineered heart tissues (EHTs) and WTC EHTs. Systolic (FIGS.5A and 5B) and diastolic (FIGS.5C and 5D) measures of contractility under spontaneous (FIGS.5A and 5C) and paced (FIGS.5B and 5D) beating conditions are shown for atrial EHTs. Systolic (FIGS.5E and 5F) and diastolic (FIGS.5G and 5H) measures of contractility under spontaneous (FIGS.5E and 5G) and paced (FIGS.5F and 5H) beating conditions are shown for ventricular EHTs. FIGS. 5I-5J show transcript levels of two important genes in contractile function regulation, TTN (FIG.5I) and ACTN (FIG.5J).

[0019] FIGS.6A-6E show expression of the YAP signaling cascade in iPSC-derived atrial cells homozygous for the rs766868752 mutation (SYNPO2L Mut+ / +), compared to control wild-type isogenic cells (WTC). LATS1 transcript levels (FIG. 6A), LATS2 transcript levels (FIG.6B), LATS2 protein levels (FIG.6C), YAP protein levels (FIG.6D), and phosphor-YAP protein levels (FIG.6E) are shown.

[0020] FIG. 7 provides RNA-seq data showing differences in transcript levels of downstream targets of YAP in iPSC-derived atrial cells homozygous for the rs766868752Attorney Docket No. TENA-054 / 01WO 334682-2486 mutation (SYNPO2L Mut+ / +), compared to control wild-type isogenic cells (WTC).

[0021] FIGS. 8A-8B show AAV expression cassettes for AAV:SYNPO2L_A and AAV:No ORF (FIG.8A) and quantification of SYNPO2L_A expression in iPSC-derived atrial cells homozygous for the rs766868752 mutation (SYNPO2L Mut+ / +) or wild-type isogenic cells (WTC) transduced with AAV:No ORF or AAV:SYNPO2L_A. In the graph, bars appear in the following order from left to right: WTC + AAV:No ORF (1); SYNPO2L Mut+ / ++ AAV:No ORF (2); SYNPO2L Mut+ / ++ AAV:SYNPO2L_A (3).

[0022] FIGS.9A-9D show electrophysiological function in iPSC-derived atrial cells homozygous for the rs766868752 mutation (SYNPO2L Mut+ / +) or wild-type isogenic cells (WTC) transduced with AAV:No ORF or AAV:SYNPO2L_A. FIG. 9A shows representative action potential traces, FIG. 9B shows APDC90, FIG. 9C shows field potential spike amplitudes, and FIG.9D shows field potential spike slopes. In the bar graphs, bars appear in the following order from left to right: WTC + AAV:No ORF (1); SYNPO2L Mut+ / ++ AAV:No ORF (2); SYNPO2L Mut+ / ++ AAV:SYNPO2L_A (3).

[0023] FIGS. 10A-10G show contractility in engineered heart tissues (EHTs) homozygous for the rs766868752 mutation (SYNPO2L Mut+ / +) or wild-type isogenic control EHTs (WTC) transduced with AAV:No ORF or AAV:SYNPO2L_A. Systolic (FIGS. 10A-10D) and diastolic (FIGS.10E-10G) measures of contractility are shown. In the bar graphs, bars appear in the following order from left to right: WTC + AAV:No ORF (1); SYNPO2L Mut+ / ++ AAV:No ORF (2); SYNPO2L Mut+ / ++ AAV:SYNPO2L_A (3).

[0024] FIG.11 shows that a common variant, rs3740293, is a splice quantitative trait locus (sQTL) in which the C allele of rs3740293 is associated with decreased expression of the B isoform in atrial heart muscle.

[0025] FIG.12 shows that carriers of another common variant, rs60632610, which is closely linked to rs3740293 (R2=0.95) and associated with decreased isoform B, have reduced risk of developing atrial fibrillation after ischemic heart disease among individuals in the UK Biobank. DETAILED DESCRIPTION

[0026] The present technology provides cardioprotective compositions and methods for preventing and / or treating heart diseases (e.g., atrial fibrillation), for example, through increasing levels of the synaptopodin 2 like (SYNPO2L) gene, isoformAttorney Docket No. TENA-054 / 01WO 334682-2486 A (SYNPO2LA), in cardiac cells in a subject. In particular, the present technology provides transgenes, expression cassettes, vectors, recombinant AAV (rAAV) viral genomes, rAAV viruses or virions, and pharmaceutical compositions for overexpressing SYNPO2LA in a cardiac cell in a subject, as well as methods of using the same in the prevention or treatment atrial fibrillation.

[0027] While the present disclosure is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the present disclosure is to be considered as an exemplification of the invention and is not intended to limit the invention to the specific embodiments illustrated. Headings are provided for convenience only and are not to be construed to limit the invention in any manner. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.

[0028] The use of numerical values in the various quantitative values specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about.” It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about.” It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios, such as about 2, about 3, and about 4, and sub-ranges, such as about 10 to about 50, about 20 to about 100, and so forth. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0029] All publications disclosed herein are incorporated by reference in their entirety. To the extent any materials incorporated by reference conflict with the present disclosure, the present disclosure controls.Attorney Docket No. TENA-054 / 01WO 334682-2486 Definitions

[0030] Generally, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise specified, each of the following terms has the meaning set forth in this section.

[0031] The indefinite articles “a” and “an” denote at least one of the associated nouns and are used interchangeably with the terms “at least one” and “one or more.” For example, the phrase “a module” means at least one module, or one or more modules.

[0032] The conjunctions “or” and “and / or” are used interchangeably.

[0033] The term “about,” as used herein when referring to a measurable value, such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0034] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises,” “comprising”, or equivalents such as “have,” “has,” “having,” “contain,” “contains,” “containing,” “include,” “includes,” or “including” will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0035] The term “AAV” is an abbreviation for adeno-associated virus. The term covers all subtypes of AAV, except where a subtype is indicated, and to both naturally occurring and recombinant forms. The abbreviation “rAAV” refers to recombinant adeno- associated virus. “AAV5” refers to AAV subtype 5. “AAV9” refers to AAV subtype 9. The genomic sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits may be found in the literature or in public databases such as GenBank. An “AAV vector” or “rAAV vector” is used in the art to refer either to the DNA packaged into in the rAAV virion or to the rAAV virion itself, depending on context. As used herein, unless otherwise apparent from context, rAAV vector refers to a nucleic acid (typically a plasmid) comprising a polynucleotide sequence capable of being packaged into an rAAV virion, but with the capsid or other proteins of the rAAV virion. Generally, an rAAV vector comprises a heterologous polynucleotide sequence (i.e., a polynucleotide not of AAV origin) and oneAttorney Docket No. TENA-054 / 01WO 334682-2486 or two AAV ITRs flanking the heterologous polynucleotide sequence. An “AAV particle” refers to an extracellular viral particle including at least one viral capsid protein (e.g., VP1) and an encapsidated AAV vector (or fragment thereof), including the capsid proteins.

[0036] The term “administering” to a subject is a procedure by which one or more delivery agents, together or separately, are introduced into or applied onto a subject such that target cells which are present in the subject are eventually contacted with the agent.

[0037] For brevity and clarity, the disclosure refers to “capsid protein” or “capsid proteins” of AAV. Those skilled in the art understand that such references refer to VP1, VP2, or VP3, or combinations thereof. As in wild-type AAV and most recombinant expression systems VP1, VP2, and VP3 are expressed from the same open reading frame, engineering of the sequence that encodes VP3 inevitably alters the sequences of the C-terminal domain of VP1 and VP2. One may also express the capsid proteins from different open reading frames, in which case the capsid of the resulting rAAV virion could contain a mixture of wild-type and engineered capsid proteins, and mixtures of different engineered capsid proteins.

[0038] The term “cardiomyopathy” refers to the deterioration of the function of the myocardium (i.e., the actual heart muscle) for any reason. Subjects with cardiomyopathy are often at risk of arrhythmia or sudden cardiac death or both. The term “hypertrophic cardiomyopathy” refers to a disease of the heart and myocardium in which a portion of the myocardium is hypertrophied. The term “familial hypertrophic cardiomyopathy” refers to a genetic disorder characterized by increased growth (i.e., hypertrophy) in thickness of the wall of the left ventricle.

[0039] A “clinically effective amount,” “clinically effective concentration,” or “clinically effective dose” refers to a concentration or dose of a peptide, composition, or pharmaceutical composition that is shown to be effective in clinical trials or is predicted to be effective based on early phase or pre-clinical trials. In some embodiments, a “clinically effective amount” is the same as a “therapeutically effective amount.” In some embodiments, a “clinically effective amount” is higher or lower than a “therapeutically effective amount.” Further, the effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the subject’s response to treatment. Various factors can influence the actual effective amount used for a particular application.Attorney Docket No. TENA-054 / 01WO 334682-2486 For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in the actual effective amount administered.

[0040] The term “construct” refers to any polynucleotide that contains a recombinant nucleic acid molecule. A construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. A “vector” is a nucleic acid molecule that is capable of introducing a specific nucleic acid sequence into a cell or into another nucleic acid sequence, or as a means of transporting another nucleic acid molecule. Vectors may be, for example, plasmids, cosmids, viruses, an RNA vector, or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi- synthetic, or synthetic nucleic acid molecules. Exemplary vectors are those capable of autonomous replication (episomal vector), capable of delivering a polynucleotide to a cell genome (e.g., viral vector), or capable of expressing nucleic acid molecules to which they are linked (expression vectors).

[0041] The term “delivery”, which is used interchangeably with “transduction,” refers to the process by which exogenous nucleic acid molecules are transferred into a cell such that they are located inside the cell. Delivery of nucleic acids is a distinct process from expression of nucleic acids.

[0042] The term “expression” refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).

[0043] The term “expression cassette” or “expression construct” refers to a DNA polynucleotide sequence operably linked to a promoter.

[0044] The term “gene therapy” involves the transfer of heterologous DNA to cells of a mammal, particularly a human, with a disorder or conditions for which therapy or diagnosis is sought. The DNA is introduced into the selected target cells in a manner such that the heterologous DNA is expressed, and a therapeutic product encoded thereby is produced. Alternatively, the heterologous DNA may in some manner mediate expression of DNA that encodes the therapeutic product; it may encode a product, suchAttorney Docket No. TENA-054 / 01WO 334682-2486 as a peptide or RNA that in some manner mediates, directly or indirectly, expression of a therapeutic product. Gene therapy may also be used to deliver nucleic acid encoding a gene product to replace a defective gene or supplement a gene product produced by the mammal or the cell in which it is introduced. The introduced nucleic acid may encode a therapeutic gene product that is not normally produced in the mammalian host or that is not produced in therapeutically effective amounts or at a therapeutically useful time. The heterologous DNA encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host to enhance or otherwise alter the product or expression thereof.

[0045] The term “host cell” as used herein refers to a cell or microorganism targeted for genetic modification by introduction of a construct or vector carrying a nucleotide sequence for expression of a protein or polypeptide of interest.

[0046] The term “modified” refers to a substance or compound (e.g., a cell, a polynucleotide sequence, and / or a polypeptide sequence) that has been altered or changed as compared to the corresponding unmodified substance or compound.

[0047] The term “nucleic acid” or “polynucleotide” refers to a polymeric compound including covalently linked nucleotides comprising natural subunits (e.g., purine or pyrimidine bases). Purine bases include adenine and guanine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single- or double-stranded. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence.

[0048] The term “operably linked” or “operatively linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a polynucleotide sequence if the promoter affects the transcription or expression of the polynucleotide sequence.

[0049] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length, though a number of amino acid residues may be specified. Polypeptides may include amino acid residues including natural and / or non-natural amino acid residues. The termsAttorney Docket No. TENA-054 / 01WO 334682-2486 also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0050] The term “promoter” as used herein refers a polynucleotide sequence that has one or more recognition site(s) to which an RNA polymerase binds, such that in a host or target cell, an RNA polymerase may initiate and transcribe a polynucleotide sequence “downstream” of the promoter into an RNA. Similarly stated, a “promoter” is operably linked or operatively linked to a polynucleotide sequence if in a host or target cell in which the promoter is active, an RNA polymerase initiates transcription of the polynucleotide at a transcription state site. Promoters operative in mammalian cells generally comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and / or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide.

[0051] The term “recombinant” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature, or that the polynucleotide is assembled from synthetic oligonucleotides. A “recombinant” protein is a protein produced from a recombinant polypeptide. A recombinant virion is a virion that comprises a recombinant polynucleotide and / or a recombinant protein, e.g., a recombinant capsid protein.

[0052] The term “sequence identity” or “identity” when referring to a polynucleotide or polypeptide sequence refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences that are the same, and in the same relative position. As such one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. Methods of sequence alignment for comparison and determination of percent sequence identity is well known in the art. Optimal alignment ofAttorney Docket No. TENA-054 / 01WO 334682-2486 sequences for comparison can be conducted, e.g., by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and 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), by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology (2003)), by use of algorithms know in the art including the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977); and Altschul et al., J. Mol. Biol.215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In some embodiments, the determination of the percentage of sequence identity may take place after a local alignment. Such alignments are well known in the art, for instance, the service EMBOSS Matcher identifies local similarities between two sequences using an algorithm based on the LALIGN application, version 2.0u4. In an example, the identity between two nucleic acid sequences may be calculated using the service Matcher (EMBOSS) set to the default parameters, e.g., matrix (DNAfull), gap open (16), gap extend (4), alternative matches (1).

[0053] The term “subject” refers to a mammalian subject, preferably a human. A “subject in need thereof” refers to a subject who has been diagnosed with a cardiac disease (e.g., cardiomyopathy) or is at an elevated risk of developing the disease. The phrases “subject” and “patient” are used interchangeably herein.

[0054] A “therapeutically effective amount” as used herein is an amount that produces a desired effect in a subject for an indication, condition, disease, or disorder. In certain embodiments, the therapeutically effective amount is an amount that yields maximum therapeutic effect. In other embodiments, the therapeutically effective amount yields a therapeutic effect that is less than the maximum therapeutic effect. For example, a therapeutically effective amount may be an amount that produces a therapeutic effect while avoiding one or more side effects associated with a dosage that yields maximum therapeutic effect. A therapeutically effective amount for a particular composition will vary based on a variety of factors, including, but not limited to, the characteristics of the therapeutic composition (e.g., activity, pharmacokinetics, pharmacodynamics, and bioavailability); the physiological condition of the subject (e.g., age, body weight, sex,Attorney Docket No. TENA-054 / 01WO 334682-2486 disease type and stage, medical history, general physical condition, responsiveness to a given dosage, and other present medications); the nature of any pharmaceutically acceptable carriers, excipients, and preservatives in the composition; and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely, by monitoring a subject’s response to administration of the therapeutic composition and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy, 21stEdition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0055] The term “transgene” refers to a nucleic acid sequence encoding a protein or RNA (e.g., a therapeutic protein), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal’s genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.

[0056] The terms “treat,” “treating,” and “treatment,” as used herein, refer to acting upon a disease, disorder, or condition with an agent to reduce or ameliorate harmful or any other undesired effects of the disease, disorder, or condition and / or its symptoms. In some embodiments, “treat,” “treating,” and “treatment” refer to reducing at least one symptom of the disease.

[0057] The terms “prevent”, “preventing”, or “prevention” as used herein refer to complete prevention of the symptoms of a disease, a delay in onset of the symptoms of a disease, or a lessening in the severity of subsequently developed disease symptoms when the agent is administered prior to the onset of one or more disease symptoms.

[0058] The term “upstream” refers to a portion of a polynucleotide that is, with reference to a transcription start site (TSS), 5’ to the TSS on the sense strand (or coding strand) of the polynucleotide; and 3’ to the TSS on the antisense strand of the polynucleotide. The term “downstream” refers to a portion of a polynucleotide that is, with reference to a TSS, 3’ to TSS on the sense strand (or coding strand) of theAttorney Docket No. TENA-054 / 01WO 334682-2486 polynucleotide; and 5’ to the TSS on the antisense strand of the polynucleotide. Thus, a deletion from the upstream end of a promoter is a deletion of one or more base pairs in the non-transcribed region of the polynucleotide, 5’ to the TSS on the sense strand (or equivalently, 3’ to the TSS on the antisense strand). A deletion from the downstream end of a promoter is a deletion of one or more base pairs in the transcribed region of the polynucleotide, 3’ to the TSS on the sense strand (or equivalently, 5’ to the TSS on the antisense strand).

[0059] The term “variant” refers to a protein or nucleic acid having one or more genetic changes (e.g., insertions, deletions, substitutions, or the like) that returns all or substantially all of the functions of the reference protein or nucleic acid. For example, a variant of a therapeutic protein retains the same or substantially the same activity and / or provides the same or substantially the same therapeutic benefit to a subject in need thereof. A variant of a promoter sequence retains the ability to initiate transcription at the same or substantially the same level as the reference promoter, and retains the same or substantially the same cell type specificity. In particular embodiments, polynucleotides variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence. In particular embodiments, protein variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence.

[0060] A “vector” refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.

[0061] The term “wild-type” or “WT” refers to the naturally-occurring polynucleotideAttorney Docket No. TENA-054 / 01WO 334682-2486 sequence encoding a protein, or a portion thereof, or protein sequence, or portion thereof, respectively, as it normally exists in vivo in a normal or healthy subject. Cardioprotective Transgenes, Expression Cassettes, Vectors, Virions, and Compositions

[0062] In some aspects, provided are cardioprotective transgenes and / or gene products suitable for use in a method of treating a heart disease, e.g., atrial fibrillation. In some embodiments, the cardioprotective gene is SYNPO2L, or a functional homolog or variant thereof. SYNPO2L is mostly expressed in cardiac cells (cardiomyocytes). There are at least two isoforms of SYNPO2L: the longer isoform of SYNPO2L, known as the adult isoform (SYNPO2LA); and the shorter isoform of SYNPO2L, known as the fetal isoform (SYNPO2LB). In some embodiments, the cardioprotective gene is SYNPO2LA, or a functional homolog or variant thereof. SYNPO2L gene information and exemplary sequences are provided in Tables 1A and 1B below. Table 1A. SYNPO2L gene informationTable 1B. Exemplary SYNPO2L sequencesAttorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486

[0063] In some embodiments, provided is a polynucleotide encoding a SYNPO2LA protein (or a mutant, variant, or fragment thereof). In some embodiments, the polynucleotide comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 1, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the SYNPO2LA protein encoded by the polynucleotide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2.

[0064] In some embodiments, provided is an expression cassette and / or a vector comprising a polynucleotide encoding a SYNPO2LA protein (or a mutant, variant, or fragment thereof) as described herein. In some embodiments, the expression cassette and / or vector comprises a polynucleotide encoding a SYNPO2LA protein (or a mutant, variant, or fragment thereof), operatively linked to a promoter, for example, a cardiac- specific promoter.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0065] The polynucleotides, expression cassettes, and / or vectors contemplated herein may be combined with other sequences, such as promoters, enhancers, untranslated regions (UTRs), introns, signal sequences, Kozak sequences, polyadenylation (poly(A)) signals, post-transcriptional regulatory elements, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, polynucleotides encoding self-cleaving polypeptides, epitope tags, and / or any other regulatory elements as disclosed elsewhere herein or as known in the art. In some embodiments, the polynucleotides, expression cassettes, and / or vectors described herein may also contain a ribosome binding site for translation initiation, a transcription terminator, and / or polynucleotide sequences for amplifying expression. The expression cassette may be flanked by one or more inverted terminal repeats (ITRs). The ITRs in an expression cassette serve as markers used for viral packaging of the expression cassette. The expression cassette can be integrated into the host cell genome, thereby expressing the transgene within a host cell.

[0066] As used herein, the term “regulatory element” refers to those non-translated regions of the vector (e.g., origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Kozak sequence), introns, poly(A) sequences, 5′ and 3′ untranslated regions) which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. The transcriptional regulatory element may be functional in either a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., bacterial or archaeal cell). In some embodiments, a polynucleotide sequence described herein is operably linked to multiple control elements that allow expression of the polynucleotide in both prokaryotic and eukaryotic cells. Promoters

[0067] The term “promoter” as used herein refers to a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning strongly active in a wide range of cells, tissues, and species or cell-type specific, tissue-specific, or species specific. Examples of ubiquitous promoters include the CAG promoter and CMV promoter. Promoters may be “constitutive,” meaning continually active, orAttorney Docket No. TENA-054 / 01WO 334682-2486 “inducible,” meaning the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors. Also included in the nucleic acid constructs or vectors of the invention are enhancer sequences that may or may not be contiguous with the promoter sequence. Enhancer sequences influence promoter-dependent gene expression and may be located in the 5ʹ or 3ʹ regions of the native gene.

[0068] In some embodiments, the promoter driving SYNPO2LA expression is any promoter suitable for protein expression described herein or known in the art. In some embodiments, the promoter is a constitutive promoter. As used herein, a “constitutive promoter” is one wherein the level of expression does not vary from one cell type compared to a different cell type. Suitable constitutive promoters include elongation factor 1 alpha (EF1α) promoter, cytomegalovirus (CMV) immediate-early promoter, simian vacuolating virus 40 (SV40) early promoter, spleen focus-forming virus (SFFV) promoter, phosphoglycerate kinase (PGK) promoter, human beta actin promoter, polyubiquitin C gene (UBC) promoter, and CAG promoter.

[0069] In some embodiments, the promoter is a cardiac-specific promoter described herein or known in the art. In some embodiments, the promoter is a cardiomyocyte- specific promoter. A “cardiac-specific” or “cardiomyocyte-specific” promoter as used herein specifies a promoter whose activity in cardiac cells or cardiomyocytes is at least 2-fold higher than in any other non-cardiac cell type. Preferably, a cardiac-specific or cardiomyocyte-specific promoter suitable for the present technology has an activity in cardiac cells or cardiomyocytes which is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 50-fold higher compared to its activity in a non-cardiac cell type. Examples of cardiac-specific or cardiomyocyte-specific promoters include, but are not limited to, alpha myosin heavy chain promoter, myosin light chain 2v promoter, alpha-cardiac actin promoter, alpha-tropomyosin promoter, cardiac troponin C promoter, cardiac troponin I promoter, cardiac myosin-binding protein C promoter, and sarco / endoplasmic reticulum Ca2+ATPase (SERCA) promoter (e.g., isoform 2 of SERCA2). Table 2A. Exemplary cardiac-specific promoter sequencesAttorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486

[0070] In some embodiments, the promoter is a cardiac troponin T (TNNT2) promoter, for example, a human TNNT2 promoter, a chicken TNNT2 promoter, a mouse TNNT2 promoter, or a chimeric or variant of any of the foregoing. In some embodiments, the TNNT2 promoter is modified from the corresponding wild-type promoter, e.g., by deletion, insertion, or substitution of polynucleotides. In some embodiments, the TNNT2 promoter is a modified human TNNT2 promoter (e.g., a truncated human TNNT2 promoter) as described herein. Illustrative polynucleotide sequences of TNNT2 promoter are shown in Table 2A. The transcription start site (TSS) of certain TNNT2 promotersAttorney Docket No. TENA-054 / 01WO 334682-2486 are bolded and underlined. As used herein, the term “transcription start site” or “TSS” refers to the first base pair transcribed by an RNA polymerase when the RNA polymerase initiates transcription. A TSS is different from the start codon (canonically, ATG), which must be downstream of the TSS in the transcribed region of the polynucleotide. The location of a TSS can be determined experimentally or by prediction using any of various prediction algorithms. Annotated TSSs are available from the Eukaryotic Promoter Database and the UCSC Genome Browser. As used herein, the TSS for TNNT2 is defined to be the sequence identified by the C at the 5’ end of the motif identified by dbTSS: CTCCATC.

[0071] Additional examples of TNNT2 promoters can be found in WO2021 / 163357, WO2023 / 283649, and / or U.S. Patent No. 11,129,908, the entire contents of each of which are incorporated by reference herein.

[0072] In some embodiments, the promoter is a human troponin T (HuTNNT2) promoter or a chimeric or variant thereof. In some embodiments, the promoter comprises, consists of, or consists essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 5-8, or a nucleotide sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 5-8.

[0073] In some embodiments, the promoter is a modified TNNT2 promoter, e.g., a promoter that comprises a polynucleotide sequence of at least 200 base pairs that comprises one or more continuous or discontinuous polynucleotide segments each sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a corresponding segment of the HuTNNT2 600 segment provided in Table 2A as SEQ ID NO: 5. As it is a “promoter,” the modified TNNT2 promoter must be capable of promoting initiation of transcription by an RNA polymerase in a host or target cell at or near a TSS within the promoter (i.e., at or near the TTS of TNNT2 as defined herein) or, if the endogenous TSS of TNNT2 is not present in the modified TNNT2 promoter then at a heterologous TSS at most 100 base pairs downstream (3’ on the sense strand) to the downstream (3’) end of the modified TNNT2 promoter. Similarly stated, the modified TNNT2 promoter may comprise only sequences upstream of the TSS of HuTNNT2600 or more comprise the TSS of TNNT2-600.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0074] In some embodiments, the promoter is a modified TNNT2 promoter comprising a polynucleotide sequence of between about 300 to about 500 base pairs, between about 350 to about 500 base pairs, between about 350 to about 450 bp, between about 400 to about 450 base pairs, or between about 375 to about 425 base pairs in length. In some embodiments, the modified TNNT2 promoter comprises a polynucleotide sequence of between about 350 base pairs to about 450 base pairs, between about 375 base pairs to about 425 base pairs, between about 375 base pairs to about 400 base pairs, between about 375 base pairs to about 425 base pairs, between about 400 base pairs to about 425 base pairs, or between about 400 base pairs to about 450 base pairs. In some embodiments, the modified TNNT2 promoter comprises a polynucleotide sequence of about 400 base pairs.

[0075] In some embodiments, the promoter has the same cell-type specificity as a native troponin T promoter of about 600 bp, for example, the reference HuTNNT2600 promoter (SEQ ID NO: 5) having about 600 base pairs.

[0076] In some embodiments, the promoter is a modified TNNT2 promoter comprising between 300 bp and 500 bp of SEQ ID NO: 5. For instance, the modified TNNT2 promoter may comprise SEQ ID NO: 6, 7, or 8. In some examples, the 300 bp- 500 bp sequence may be linked to further polynucleotide sequences but may not be linked to additional sequences derived from SEQ ID NO: 5. For example, in an embodiment, the modified TNNT2 promoter may include not more than 500 bp of SEQ ID NO: 5 but may include additional unrelated polynucleotide sequences. In another example, the modified TNNT2 promoter may include SEQ ID NO: 6, 7, or 8 and no additional sequences derived from SEQ ID NO: 5 but may include additional unrelated polynucleotide sequences.

[0077] In some embodiments, the TNNT2 promoter is modified by deletion of polynucleotides. A modification may include one, two, three or more internal deletions. Each deletion may be a deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to the reference HuTNNT2600 promoter (SEQ ID NO: 5) having about 600 base pairs.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0078] In some embodiments, the TNNT2 promoter is modified by the deletion of polynucleotides from the upstream end of the promoter with respect to the reference HuTNNT2 600 promoter (SEQ ID NO: 5) having about 600 base pairs. A modification may include the deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs from the upstream end of the promoter with respect to the reference HuTNNT2600 promoter (SEQ ID NO: 5) having about 600 base pairs. In some embodiments, the modification is a 200 base pair deletion from the upstream end of the promoter with respect to the reference HuTNNT2600 promoter (SEQ ID NO: 5) having about 600 base pairs.

[0079] In some embodiments, the TNNT2 promoter is modified by the deletion of polynucleotides from the downstream end of the promoter with respect to the reference HuTNNT2 600 promoter (SEQ ID NO: 5) having about 600 base pairs. A modification may include the deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs from the downstream end of the promoter with respect to the reference HuTNNT2600 promoter (SEQ ID NO: 5) having about 600 base pairs.

[0080] In some embodiments, the TNNT2 promoter is modified by an internal deletion of polynucleotides. A modification may include the internal deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 30 base pairs, 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to the reference HuTNNT2600 promoter (SEQ ID NO: 5) having about 600 base pairs.

[0081] In some embodiments, the TNNT2 promoter is modified by the insertion of polynucleotides. A modification may include the insertion of 1 base pair, 2 base pairs, 3Attorney Docket No. TENA-054 / 01WO 334682-2486 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 35 base pairs, 40 base pairs, 45 base pairs, 50 base pairs, 55 base pairs, 60 base pairs, 65 base pairs, 70 base pairs, 75 base pairs, 80, base pairs, 85 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to the reference HuTNNT2600 promoter (SEQ ID NO: 5) having about 600 base pairs.

[0082] In some embodiments, the TNNT2 promoter is modified by the substitution of polynucleotides. A modification may include the substitution of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 6 base pairs, 7 base pairs, 8 base pairs, 9 base pairs, or 10 base pairs with respect to the reference HuTNNT2600 promoter (SEQ ID NO: 5) having about 600 base pairs.

[0083] In some embodiments, the promoter is a mouse troponin T (MsTNNT2) promoter or a chimeric or variant thereof. In some embodiments, the promoter comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 9, or a nucleotide sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9.

[0084] In some embodiments, the promoter is a chicken troponin T (ChTNNT2) promoter or a chimeric or variant thereof. In some embodiments, the promoter comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 10, or a nucleotide sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10.

[0085] In some embodiments, the promoter is a chimeric variant derived from the human, mouse, or chicken TNNT2 promoter. A chimeric promoter can be generated by combining fragments from the TNNT2 promoter to sequences from cardiac-specific promoters or ubiquitous or core promoters. In some embodiments, the promoter comprises, consists of, or consists essentially of a nucleotide sequence set forth in any one of SEQ ID NOs: 11-16, or a nucleotide sequence that shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 11-16.Attorney Docket No. TENA-054 / 01WO 334682-2486 Enhancers

[0086] The term “enhancer” refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and / or other enhancer elements. The term “enhancer” further refers to a DNA sequence that directs the binding of transcriptional regulatory proteins (e.g., transcriptional machinery) and RNA polymerase, and thereby promotes RNA synthesis. An enhancer may overlap with a promoter or be upstream or downstream of the promoter.

[0087] In some embodiments, the expression cassette and / or vector further comprises one or more enhancers. The one or more enhancers can be operably linked to the promoter and modulate the expression of a transgene operably linked to the promoter. The presence of an enhancer can modulate transgene expression by, for example, increasing expression or decreasing expression. An enhancer can modulate transgene expression by, for example, increasing expression levels in a desired cell type, for example, a cardiac cell. An enhancer can modulate transgene expression by, for example, decreasing expression levels in an “off-target” cell type, or a cell type in which expression is not desired. In some embodiments, the promoter is a TNNT2 promoter and comprises one or more enhancers. For example, a ACTC1 cardiac enhancer can be linked to a human TNNT2 promoter. In some embodiments, the promoter is a TNNT2 promoter and comprises no enhancer. In some embodiments, the vector comprises an enhancer that is operably linked to another enhancer. For example, a ACTC1 cardiac enhancer can be operably linked to an αMHC enhancer. In some embodiments, the expression cassette comprises an enhancer that is operably linked to a promoter and operably linked to another enhancer. Exemplary enhancers are provided in Table 2B below. Table 2B. Exemplary enhancer sequencesAttorney Docket No. TENA-054 / 01WO 334682-2486

[0088] In some embodiments, the enhancer comprises an ACTC1 cardiac enhancer (ACTC1e). In some embodiments, the enhancer comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 17, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17.

[0089] In some embodiments, the enhancer comprises an αMHC enhancer (αMHCe). In some embodiments, the enhancer comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 18, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 18. Introns

[0090] In some embodiments, the expression cassette and / or vector further comprises one or more intron sequences, for example, a synthetic or chimeric intron sequence. The intron sequence can be used to adjust the length (i.e., size) of the expression cassette for improving recombinant AAV packaging. The intron sequence can also be used to improve the efficiency of transgene expression (i.e., mRNA production or transcription) in a host cell containing the expression cassette from the vector. Exemplary intron sequences are provided in Table 2C below. Table 2C. Exemplary intron sequencesAttorney Docket No. TENA-054 / 01WO 334682-2486

[0091] In some embodiments, the intron comprises an CMV intron (CMVint). In some embodiments, the intron comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 19, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 19.

[0092] In some embodiments, the intron comprises a chimeric intron (Chimint). In some embodiments, the intron comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 20, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 20. WPRE sequences and other post-transcriptional elements

[0093] In some embodiments, the expression cassette and / or vector further comprises one or more post-transcriptional regulatory elements, for example, a woodchuck hepatitis virus post-transcriptional element (WPRE). The WPRE sequence can be inserted, for example, proximal to on the 3’ end of a transgene in a viral vector to, for example, optimize gene expression in a viral vector. In some embodiments, the WPRE comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 21, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 21. In some embodiments, the expression cassette and / or vector does not comprise a WPRE. Table 2D. Exemplary WPRE sequencesPoly(A) sequencesAttorney Docket No. TENA-054 / 01WO 334682-2486

[0094] In some embodiments, the expression cassette and / or vector further comprises one or more poly(A) sequences. The term “poly(A) sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by addition of a poly(A) tail to the 3′ end of the coding sequence and thus, contribute to increased translational efficiency. Cleavage and polyadenylation are directed by a poly(A) sequence in the RNA. The core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage-polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5’ cleavage product. In someembodiments, the core poly(A) sequence is an ideal poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). Non-limiting examples of poly(A) sequences include SV40 poly(A) sequence, bovine growth hormone (BGH) poly(A) sequence, rabbit β-globin poly(A) sequence (rβgpA), variants thereof, and other suitable heterologous or endogenous poly(A) sequences known in the art. Exemplary poly(A) sequences are provided in Table 2E below. Table 2E. Exemplary poly(A) sequences

[0095] In some embodiments, the poly(A) sequence comprises a synthetic poly(A) sequence. In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 22, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0096] In some embodiments, the poly(A) sequence comprises a BGH poly(A) sequence. In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 23, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 23.

[0097] In some embodiments, the poly(A) sequence comprises a SV40 poly(A) sequence. In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 24, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 24. Additional elements

[0098] In some embodiments, the expression cassette and / or vector further comprises one or more additional elements, for example, a Kozak sequence and a nuclear localization sequence (NLS). Exemplary additional regulatory sequences are provided in Table 2F below. Table 2F. Exemplary additional regulatory sequences

[0099] In some embodiments, the expression cassette and / or vector comprises a Kozak sequence comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 25, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 25.

[0100] In some embodiments, the expression cassette and / or vector comprises a SV40 NLS comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 26, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 26.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0101] In some embodiments, the expression cassette and / or vector comprises a nucleoplasmin NLS comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 27, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 27.

[0102] In some embodiments, the expression cassette and / or vector further comprises a transcription termination signal. Elements directing the efficient termination and polyadenylation of the heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. ITRs

[0103] In some embodiments, the expression cassette is flanked by AAV inverted terminal repeats (ITRs) at the 5’ and 3’ ends. ITRs function as recognition sites for replication and markers used for viral packaging of the expression cassette. ITRs form T-shaped secondary structures by two adjacent inverted repeats separated by an unpaired nucleotide. ITRs are required for packaging the expression cassette into an rAAV virion, which provide the function of expressing the transgene after a host cell is targeted by the rAAV virion. The ITRs contain tetranucleotide repeat motifs called Rep- binding elements (RBE) that act as contact points for the Rep68 / 78 proteins encoded by the rep gene. The ITRs also contain a packaging signal for genome encapsidation, which directs 3’ genomic transport into preassembled capsids by Rep proteins. Any naturally occurring or synthetically derived ITRs described herein or known in the art can be used.

[0104] In some embodiments, the ITRs flanking the expression cassette are ITRs of the same AAV serotype as the Rep protein used in making the virions described herein. For example, where a Rep protein from AAV9 is used, the transgene expression cassette used in the expression system comprises ITRs from AAV9 as well. In another example, where a Rep protein from AAV2 is used, the transgene expression cassette used in the expression system comprises ITRs from AAV2 as well. In another example, where a Rep protein from AAV5 is used, the transgene expression cassette used in the expression system comprises ITRs from AAV5 as well. The ITRs may be of the same or different serotype as the capsid protein used in packaging the virion described herein.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0105] In some embodiments, the ITRs comprise, consist of, or consist essentially of a nucleotide sequence set forth in SEQ ID NO: 28 or 29, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 28 or 29, as shown in Table 2G below. Table 2G. Exemplary ITR sequences

[0106] In some embodiments, the expression cassette is flanked by one or both of a 5’ ITR and a 3’ ITR. In some embodiments, the 5’ ITR comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 49, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 49. In some embodiments, the 3’ ITR comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 50, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 50.

[0107] In some embodiments, the expression cassette comprises a polynucleotide encoding a SYNPO2L_A polypeptide operably linked to a TNNT2 promoter or chimeric variant thereof. In some embodiments, the SYNPO2L_A polypeptide is a wild type human SYNPO2L_A polypeptide comprising a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2. In some embodiments, the SYNPO2L_A polypeptide is a wild type human SYNPO2L_A polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

[0108] In some embodiments, the polynucleotide encoding the wild type human SYNPO2L_A polypeptide is a codon-optimized polynucleotide. In some embodiments,Attorney Docket No. TENA-054 / 01WO 334682-2486 the polynucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 1.

[0109] In some embodiments, the TNNT2 promoter or chimeric variant thereof is a human TNNT2 promoter. In some embodiments, the TNNT2 promoter or chimeric variant thereof comprises a sequence sharing at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to any one of SEQ ID NOs: 5-16.

[0110] In some embodiments, the human TNNT2 promoter is a wild-type human TNNT2 promoter of 600 bp, optionally having 100% identity to SEQ ID NO: 5. In some embodiments, the human TNNT2 promoter has a sequence sharing at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to SEQ ID NO: 7. In some embodiments, the human TNNT2 promoter is a modified human TNNT2 promoter of 350 bp to 450 bp, or 350 bp to 500 bp. In some embodiments, the human TNNT2 promoter is a modified human TNNT2 promoter of about 400 bp of SEQ ID NO: 7.

[0111] In some embodiments, the expression cassette comprises a polyadenylation sequence. In some embodiments, the polyadenylation sequence is a bGH poly(A) sequence. In some embodiments, the bGH poly(a) has a sequence at least 50%, at least 75%, at least 80%, at least 95% or 100% identical to SEQ ID NO: 23. Vectors

[0112] In some embodiments, the polynucleotide encoding a SYNPO2LA protein for use in the present technology are in the form of a vector. The vector can be any viral vector or non-viral vector known in the art or described herein. In some embodiments, the vector is a viral vector. In some embodiments the viral vector is an adeno-associated virus vector (AAV), an adenoviral vector, a lentiviral vector, a retroviral vector, a herpes simplex virus vector (HSV), or a poxvirus vector.

[0113] As used herein, the term “retrovirus” or “retroviral” refers an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Retrovirus vectors are a common tool for gene delivery. Once the virus is integrated into the host genome, it is referred to as a “provirus.” The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules encoded by the virus. In some embodiments, a retroviral vector is altered so that it does not integrate into the hostAttorney Docket No. TENA-054 / 01WO 334682-2486 cell genome. Illustrative retroviruses include, but are not limited to, (1) genus gammaretrovirus, such as, Moloney murine leukemia virus (M-MuLV or MMLV), Moloney murine sarcoma virus (MoMSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), and feline leukemia virus (FLV); (2) genus spumavirus, such as, simian foamy virus; and (3) genus lentivirus, such as, human immunodeficiency virus-1 and simian immunodeficiency virus.

[0114] As used herein, the term “lentiviral” or “lentivirus” refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include but are not limited to, human immunodeficiency virus (HIV), including HIV type 1, and HIV type 2; visna-maedi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0115] In some embodiments, the viral vector is an adenoviral vector. The genetic organization of adenovirus includes an approximate 36 kb, linear, double-stranded DNA virus, which allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb.

[0116] In some embodiments, the viral vector is an AVV vector, such as an AAV vector selected from the group consisting of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rh.10, rh.20, rh.74, and a variant or chimeric AAV derived thereof. In some embodiments, the AAV expression vector is pseudotyped to enhance targeting. A pseudotyping strategy can promote gene transfer and sustain expression in a target cell type. For example, the AAV2 genome can be packaged into the capsid of another AAV serotype such as AAV5, AAV7, or AAV8, producing pseudotyped vectors such as AAV2 / 5, AAV2 / 7, and AAV2 / 8 respectively, as described in Balaji et al., J. Surg. Res. Sep. (2013) 184(1):691-698. In some embodiments, an AAV9 may be used to target expression in myofibroblast-like lineages, as described in Piras et al., Gene Therapy (2016) 23:469- 478. In some embodiments, AAV1, AAV6, or AAV9 is used, and in some embodiments, the AAV is engineered, as described in Asokari et al., Hum. Gene Ther. Nov. (2013) 24(11):906-913; Pozsgai et al., Mol. Ther. (2017) 25(4): 855-869; Kotterman, M.A. and D.V. Schaffer, Nature Reviews Genetics (2014) 15:445-451; and US20160340393A1 to Schaffer et al. In some embodiments, the viral vector is AAV engineered to increaseAttorney Docket No. TENA-054 / 01WO 334682-2486 target cell infectivity as described in US20180066285A1. In some embodiments, the vector is an AAV9 vector.

[0117] In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a naked DNA (e.g., a DNA plasmid). In some embodiments, the non-viral vector is a plasmid. In some embodiments, the non-viral vector is a liposome or lipid vector comprising plasmid DNA and a lipid solution.

[0118] In some embodiments, the vector is a recombinant vector.

[0119] In some aspects of the disclosure, a vector is used to deliver the expression cassette described herein to cardiac cells of a subject, e.g., to treat cardiomyopathy.

[0120] In some embodiments, the viral vectors described herein are replication incompetent, in that it cannot independently further replicate and package its genome. For example, when a cardiac cell is targeted with a virion, the transgene is expressed in the targeted cardiac cell, however, since the targeted cardiac cell lacks packaging and accessory function genes, the virion is not able to replicate. In some embodiments, the viral vectors described herein are replication competent.

[0121] In some embodiments, the vectors described herein are capable of being delivered to both dividing and non-dividing cells. In some embodiments, the vectors described herein are capable of being delivered to non-dividing cells. In some embodiments, the vectors described herein are capable of being delivered to dividing cells.

[0122] In some embodiments, the vectors comprising the expression cassettes described herein lead to cardiac cell-specific expression of the coding sequence(s). In some embodiments, the vectors comprising the expression cassettes described herein lead to cardiomyocyte-specific expression of the coding sequence(s). In some embodiments, the vectors comprising the expression cassettes described herein allow high expression of the coding sequence(s) in a cardiac cell (e.g., a cardiomyocyte) and low or no expression in other cells (e.g., low or no expression in liver cells, low or no expression in muscle cells except for muscle cells of the heart, low or no expression in cardiac fibroblasts). In some embodiments, the vectors comprising the expression cassettes described herein allow high expression of the coding sequence(s) in heart tissue of a subject (e.g., in human heart). In some embodiments, the vectors comprisingAttorney Docket No. TENA-054 / 01WO 334682-2486 the expression cassettes described herein allow no or low expression of the coding sequence(s) in tissues of a subject other than the heart (e.g., in liver or in muscles except those of the heart). “High” and “low” can be relative to each other, for example, the expression of a transgene in cardiac cells (e.g., cardiomyocytes) and / or heart tissue can be at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 150-fold, or 200-fold higher than its expression in other cells and tissues (e.g., liver, muscle except for the heart).

[0123] In some embodiments, the vector genome has a size of less than 6 kilobases. In some embodiments, the vector genome has a size of less than 5.6 kilobases. In some embodiments, the vector genome has a size of about, at most or less than 4.0 kilobases, 4.5 kilobases, 4.6 kilobases, 4.7 kilobases, 4.8 kilobases, 4.9 kilobases, 5 kilobases, 5.1 kilobases, 5.2 kilobases, 5.3 kilobases, 5.4 kilobases, or 5.5 kilobases. In some embodiments, the vector genome has a size of 4 kilobases to 5.2 kilobases. In some embodiments, the vector genome has a size of 4 kilobases to 5 kilobases. In some embodiments, the vector genome has a size of 4 kilobases to 4.8 kilobases. In some embodiments, the vector genome has a size of equal to or less than 4.9 kilobases. In some embodiments, the vector genome has a size of equal to or less than 4.8 kilobases. In some embodiments, the vector genome has a size of equal to or less than 4.7 kilobases. In some of these embodiments, the vector is an AAV vector, e.g., an AAV9 vector.

[0124] In some of these embodiments, the vector is an AAV vector or a variant thereof. In some of these embodiments, the vector is an AAV9 vector or a variant thereof. In some of these embodiments, the vector is an AAV5 vector or a variant thereof. In some of these embodiments, the vector is an AAV2 vector or a variant thereof.

[0125] The capsid proteins of AAV largely determine the immunogenicity and tropism of AAV vectors. In some embodiments, the AAV is an AAV subtype 9 (AAV9). In some embodiments, AAV9 is a preferred AAV vector due to its ability to transduce the heart following systemic delivery. While AAV9 can achieve moderate transduction of the heart, the majority of vector traffics to the liver. Moreover, in order to achieve therapeutic levels of transduction in the heart, relatively high systemic doses are required, potentially leading to systemic inflammation and in turn, toxicity.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0126] Methods of introducing polynucleotides into a host cell are known in the art, and any known method can be used to introduce the polynucleotides described herein into a cell. Suitable methods include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery, microfluidics delivery methods, and the like.

[0127] In some embodiments, the polynucleotide encoding a SYNPO2LA protein for use in the present technology are in the form of a recombinant AAV (rAAV) virus or virion, for example, to deliver the polynucleotide or expression cassettes described herein to cardiac cells.

[0128] In some embodiments, the AAV is any AAV known in the art or described herein. In some embodiments, the AAV is an AAV selected from the group consisting of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rh.10, rh.20, rh.74, or a chimeric or variant AAV derived therefrom. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.20, AAVrh.74, or a variant thereof.

[0129] In some embodiments, the rAAV virus or virion comprises an AAV capsid protein and an expression cassette as described herein. Capsid proteins are structural proteins that make up the assembled icosahedral packaging of the rAAV virion that contains the expression cassette. Capsid proteins are classified by the serotype. Wild- type capsid serotypes in rAAV virions can be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.20, or AAVrh.74. Engineered capsid types include chimeric capsids and mosaic capsids. Capsids are selected for rAAV virions based on their ability to transduce specific tissue or cell types.

[0130] Any capsid protein that can facilitate rAAV virion transduction into cardiac cells for delivery of a transgene, as described herein, can be used. Capsid proteins used in rAAV virions for transgene delivery to cardiac cells that result in high expression can be, for example, AAV4, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the AAVAttorney Docket No. TENA-054 / 01WO 334682-2486 capsid protein described herein is a wild-type AAV capsid protein from AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rh.10, rh.20, rh.74, or a variant thereof. In some embodiments, the AAV is AAV9 or a variant thereof. In some embodiments, the AAV is AAV5 or a variant thereof. In some embodiments, the AAV is AAV2 or a variant thereof.

[0131] Artificial capsids, such as chimeric capsids generated through combinatorial libraries, can also be used for transgene delivery to cardiac cells that results in high expression. Other capsid proteins with various features can also be used in the rAAV virions of the disclosure. AAV vectors and capsids are provided in U.S. Pat. Pub. Nos. US10011640B2; US7892809B2, US8632764B2, US8889641B2, US9475845B2, US10889833B2, US10480011B2, and US10894949B2, the entire contents of each of which are incorporated by reference herein; and Int’l Pat. Pub. Nos. WO2020198737A1, WO2019028306A2, WO2016054554A1, WO2018152333A1, WO2017106236A1, WO2008124724A1, WO2017212019A1, WO2020117898A1, WO2017192750A1, WO2020191300A1, and WO2017100671A1, the entire contents of each of which are incorporated by reference herein.

[0132] In some embodiments, the rAAV virus or virion comprises a wild-type AAV9 capsid protein or a variant thereof. Wild-type AAV9 VP1 has the amino acid sequence of SEQ ID NO: 30; wild-type AAV9 VP2 has the amino acid sequence of SEQ ID NO: 31; wild-type AAV9 VP3 has the amino acid sequence of SEQ ID NO: 32, as shown below and provided in Table 3A. The N-terminal residue of VP1, VP2, and VP3, the VR sites (e.g., VR-I, VR-II, VR-IV, VR-V, VR-VII, and VR-VIII) , as well as the last 35 amino acid positions, are indicated (in bold, and underlined) in the sequence of full-length VP1 (SEQ ID NO: 30). In some embodiments, the capsid protein comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 30-32. VP1--> (SEQ ID NO: 30) MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEP VN AADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLG LV VP2--> (SEQ ID NO: 31)Attorney Docket No. TENA-054 / 01WO 334682-2486 EEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPS GV VP3--> (SEQ ID NO: 32) GSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISN ST VR-I VR- II SGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDN NG VKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSF YC VR-IV LEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQT LK VR-V FSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMAS HK VR-VII VR-VIII EGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQ TG WVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADP PT AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEP RP IGTRYLTRNL

[0133] As labeled in AAV9 VP1 (SEQ ID NO: 30) above, the VR-I site is between amino acids 262 and 269 in the parental sequence (“NSTSGGSS”, SEQ ID NO: 103); the VR- site is between amino acids 327 and 332 in the parental sequence (“DNNGVK”, SEQ 104); the VR-IV site is between amino acids 448 and 462 in the parental sequence (“SKTINGSGQNQQTLK”, SEQ ID NO: 33); the VR-V site is between amino acids 491 and 504 in the parental sequence (“TTVTQNNNSEFAWP”, SEQ ID NO: 34); the VR-VII site is between amino acids 547 and 557 in the parental sequence (“GTGRDNVDADK”, SEQ ID NO: 35); the VR-VIII site is between amino acids 581 andAttorney Docket No. TENA-054 / 01WO 334682-2486 595 in the parental sequence (“ATNHQSAQAQAQTGW”, SEQ ID NO: 36) ; the last 35 amino acid positions are between amino acids 702 to 736 in the parental sequence (“TSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL”, SEQ ID NO: 105. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 30, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 30, excluding the VR-IV and / or VR- VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 30, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 30, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 31, excluding the VR-I, VR-II, VR- IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 31, excluding the VR-IV and / or VR-VIII site. In some embodiments, the engineered AAV9 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 30, excluding the VR-II site and / or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 31, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 31, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 31, excluding the VR-II site and / or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80%Attorney Docket No. TENA-054 / 01WO 334682-2486 (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 32, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 32, excluding the VR-IV and / or VR- VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 32, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 32, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 32, excluding the VR-II site and / or the last 32 amino acid positions.

[0134] In some embodiments, the rAAV virus or virion comprises a wild-type AAV5 capsid protein or a variant thereof. Wild-type AAV5 VP1 has the amino acid sequence of SEQ ID NO: 37; wild-type AAV5 VP2 has the amino acid sequence of SEQ ID NO: 38; wild-type AAV5 VP3 has the amino acid sequence of SEQ ID NO: 39, as shown below and provided in Table 3A. The N-terminal residue of VP1, VP2, and VP3, the VR sites (e.g., VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, as well as the last 35 amino acid positions), are indicated (in bold, and underlined) in the sequence of full-length VP1 (SEQ ID NO: 37). In some embodiments, the capsid protein comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 37-39. VP1--> (SEQ ID NO: 37) MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPV NR ADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGL VE VP2--> (SEQ ID NO: 38) VP3--> (SEQ ID NO: 39)Attorney Docket No. TENA-054 / 01WO 334682-2486 EGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAG GG VR-I GPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANA YF VR-II GYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLT ST VQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSK ML VR-IV RTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANT YK VR-V NWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENT MI VR-VII VR-VIII FNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVP GS VWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSF IT QYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

[0135] As labeled in AAV5 VP1 (SEQ ID NO: 37) above, the VR-I site is between amino acids 252 and 256 in the parental sequence (“SGSVD”, SEQ ID NO: 106); the VR- II site is between amino acids 316 and 321 in the parental sequence (“VQDSTT”, SEQ ID NO: 107); the VR-IV site is between amino acids 437 and 461 in the parental sequence (“RFVSTNNTGGVQFNKNLAGRYANTY”, SEQ ID NO: 40); the VR-V site is between amino acids 477 and 490 in the parental sequence (“LGSGVNRASVSAFA”, SEQ ID NO: 41); the VR-VII site is between amino acids 533 and 546 in the parental sequence (“PANPGTTATYLEGN”, SEQ ID NO: 42); the VR-VIII site is between amino acids 570 and 584 in the parental sequence (“ATNNQSSTTAPATGT”, SEQ ID NO: 43); the last 35 amino acid positions are between amino acids 690 and 724 in the parental sequence (“TNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL”, SEQ ID NO: 108). In someAttorney Docket No. TENA-054 / 01WO 334682-2486 embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 37, excluding the VR-IV, VR-V, VR-VII, and / or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 37, excluding the VR-IV and / or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 37, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 37, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 37, excluding the VR-II site and / or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 38, excluding the VR-IV, VR-V, VR-VII, and / or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 38, excluding the VR-IV and / or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 38, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 38, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 38, excluding the VR-II site and / or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 39, excluding the VR-IV, VR-V, VR-VII, and / or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity toAttorney Docket No. TENA-054 / 01WO 334682-2486 SEQ ID NO: 39, excluding the VR-IV and / or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 39, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 39, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 39, excluding the VR-II site and / or the last 35 amino acid positions. Table 3A. Exemplary wild-type AAV capsid protein sequencesAttorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486

[0136] In some embodiments, the engineered capsid protein is an engineered AAVrh.10 capsid protein comprising one or more amino acid substitutions and / or insertions compared to the wild-type AAVrh.10 capsid protein described herein. In some embodiments, the engineered capsid protein is an engineered AAVrh.10 capsid protein comprising an insertion peptide sequence or insertion motif compared to the wild-type AAVrh.10 capsid protein.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0137] The wild-type AAVrh.10 VP1 has the amino acid sequence of SEQ ID NO: 109; the wild-type AAVrh.10 VP2 has the amino acid sequence of SEQ ID NO: 110; the wild-type AAVrh.10 VP3 has the amino acid sequence of SEQ ID NO: 111, as shown below and provided in Table 3A. The N-terminal residue of VP1, VP2, and VP3, the variable region (VR) sites (e.g., VR-I, VR-II, VR-IV, VR-V, VR-VII and VR-VIII), as well as the last 35 amino acid positions, are indicated in bold and underlined in the sequence of full-length VP1 (SEQ ID NO: 109). In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 109. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 110. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 111. VP1--> (SEQ ID NO: 109) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEP VNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVL VP2--> (SEQ ID NO: 110) EPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPI VP3--> (SEQ ID NO: 111) GEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPT VR-I YNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLN VR-II FKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYG YLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLI VR-IV VR-V DQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFA VR-VII WTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEE VR-VIIIAttorney Docket No. TENA-054 / 01WO 334682-2486 IKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNF HPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSTNVDFAVNTDGTYSEPRPIGTRYLTRNL

[0138] As labeled in AAVrh.10 VP1 (SEQ ID NO: 112) above, the VR-I site is between amino acids 263 and 267 in the parental sequence (“NGTSG”, SEQ ID NO: 113); the VR-II site is between amino acids 328 and 333 in the parental sequence (“QNEGTK”, SEQ ID NO: 114); the VR-IV site is between amino acids 449 and 464 in the parental sequence (“SRTQSTGGTAGTQQLL”, SEQ ID NO: 115); the VR-V site is between amino acids 493 and 506 in the parental sequence (“TTLSQNNNSNFAWT”, SEQ ID NO: 116); the VR-VII site is between amino acids 549 and 559 in the parental sequence (“GAGKDNVDYSS”, SEQ ID NO: 117); the VR-VIII site is between amino acids 583 and 597 in the parental sequence (“ADNLQQQNAAPIVGA”, SEQ ID NO: 118); the last 35 amino acid positions are between amino acids 704 and 738 in the parental sequence (“TSNYYKSTNVDFAVNTDGTYSEPRPIGTRYLTRNL”, SEQ ID NO: 119). In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 109, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 109, excluding the VR-IV and / or VR-VIII site. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 109, excluding the VR-II site and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 110, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 110, excluding the VR-VI and / or VR-VIII site. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%,Attorney Docket No. TENA-054 / 01WO 334682-2486 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 110, excluding the VR-II site and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 111, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 111, excluding the VR-IV and / or VR-VIII site. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 111, excluding the VR-II site and / or the last 35 amino acid positions.

[0139] In some embodiments, the engineered capsid protein is an engineered AAVrh.74 capsid protein comprising one or more amino acid substitutions and / or insertions compared to the wild-type AAVrh.74 capsid protein described herein. In some embodiments, the engineered capsid protein is an engineered AAVrh.74 capsid protein comprising an insertion peptide sequence or insertion motif compared to the wild-type AAVrh.74 capsid protein.

[0140] The wild-type AAVrh.74 VP1 has the amino acid sequence of SEQ ID NO: 119; the wild-type AAVrh.74 VP2 has the amino acid sequence of SEQ ID NO: 120; the wild-type AAVrh.74 VP3 has the amino acid sequence of SEQ ID NO: 121, as shown below and provided in Table 3A. The N-terminal residue of VP1, VP2, and VP3, the variable region (VR) sites (e.g., VR-I, VR-II, VR-IV, VR-V, VR-VII and VR-VIII) , as well as the last 35 amino acid positions, are indicated in bold and underlined in the sequence of full-length VP1 (SEQ ID NO: 119). In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 119. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 120. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 121.Attorney Docket No. TENA-054 / 01WO 334682-2486 VP1--> (SEQ ID NO: 119) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEP VNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVL VP2--> (SEQ ID NO: 120) EPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPI VP3--> (SEQ ID NO: 121) GEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPT VR-I YNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLN VR-II FKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYG YLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLI VR-IV VR-V DQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFA VR-VII WTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEE VR-VIII IKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNF HPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

[0141] As labeled in AAVrh.74 VP1 (SEQ ID NO: 119) above, the VR-I site is between amino acids 263 and 267 in the parental sequence (“NGTSG”, SEQ ID NO: 112); the VR-II site is between amino acids 328 and 333 in the parental sequence (“QNEGTK”, SEQ ID NO: 113); the VR-IV site is between amino acids 449 and 464 in the parental sequence (“SRTQSTGGTAGTQQLL”, SEQ ID NO: 114); the VR-V site is between amino acids 493 and 506 in the parental sequence (“TTLSQNNNSNFAWT”, SEQ ID NO: 115); the VR-VII site is between amino acids 549 and 559 in the parental sequence (“GAGKDNVDYSS”, SEQ ID NO: 116); the VR-VIII site is between amino acids 583 and 597 in the parental sequence (“ADNLQQQNAAPIVGA”, SEQ ID NO: 117); the last 35 amino acid positions are between amino acids 704 and 738 in the parental sequence (“TSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL”, SEQ ID NO: 118). In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence thatAttorney Docket No. TENA-054 / 01WO 334682-2486 shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 119, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 119, excluding the VR-IV and / or VR-VIII site. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 119, excluding the VR-II site and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 120, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 120, excluding the VR-IV and / or VR-VIII site. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 120, excluding the VR-II and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 121, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and / or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 121, excluding the VR-IV and / or VR-VIII site. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 121, excluding the VR-II site and / or the last 35 amino acid positions

[0142] In some embodiments, the rAAV virus or virion comprises a chimeric capsid protein, for example, an AAV5 / AAV9 chimeric capsid protein. In some embodiments, the AAV5 / AAV9 chimeric capsid protein comprises at least 1, 2, 3, 4, 5 or more polypeptide segments each that are derived from AAV9 capsid protein and from AAV5 capsid protein.Attorney Docket No. TENA-054 / 01WO 334682-2486 In some embodiments, at least one polypeptide segment is derived from the AAV5 capsid protein and at least one polypeptide segment is derived from the AAV9 capsid protein.

[0143] In some embodiments, the rAAV virus or virion comprises a combinatory capsid protein. As used herein, “combinatory capsid protein” refers to a AAV5 / AAV9 chimeric capsid protein, which further comprises amino acid variations with respect to the chimeric parental sequence at one or more sites. In some embodiments, the one or more sites of the chimeric parental sequence are selected from those equivalent to the VR-IV site, the VR-V site, the VR-VII site, and the VR-VIII site of the corresponding wild- type capsid protein.

[0144] In some embodiments, the rAAV virus or virion comprises an engineered capsid protein. Engineered capsid proteins can be derived from a parental, e.g., wild- type, capsid and include, for example, a variant polypeptide sequence with respect to a parental capsid sequence at one or more sites. For example, variant polypeptide sequences of the parental capsid can occur at the VR-IV site, VR-V site, VR-VII site and / or VR-VIII site. In some embodiments, the engineered capsid protein has a substitution or insertion at the VR-IV region of the capsid protein (e.g., of wild-type AAV9). In some embodiments, the engineered capsid protein has a substitution or insertion at the VR-V region of the capsid protein (e.g., of wild-type AAV9). In some embodiments, the engineered capsid protein has a substitution or insertion at the VR-VII region of the capsid protein (e.g., of wild-type AAV9). In some embodiments, the engineered capsid protein has a substitution or insertion at the VR-VIII region of the capsid protein (e.g., of wild-type AAV9). In some embodiments, the engineered capsid protein has a substitution or insertion at the VR-IV region and the VR-VIII region of the capsid protein (e.g., of wild- type AAV9). Exemplary variant AAV9 capsid proteins (e.g., comprising one or more substitutions or insertions) are described in WO2021 / 163357, WO2021 / 216456, and U.S. Patent No.11,129,908, the entire contents of each of which are incorporated by reference herein. In some embodiments, the present disclosure provides a rAAV capsid protein disclosed in WO2021 / 163357 as CR9-01, and this disclosure is specifically incorporated by reference herein in its entirety.

[0145] In some embodiments, the rAAV virus or virion comprises an engineered AAV9 capsid protein comprising a variant polypeptide sequence. In some embodiments, the variant polypeptide sequence comprises one, two, three, four, five, six, seven, eight,Attorney Docket No. TENA-054 / 01WO 334682-2486 nine, ten, eleven, twelve, thirteen, or fourteen substitutions at the VR-VIII site relative to the parental sequence at the VR-VIII site. In some embodiments, the variant polypeptide sequence comprises at least six substitutions at the VR-VIII site relative to the parental sequence at the VR-VIII site. In some embodiments, the variant polypeptide sequence comprises at least eight substitutions at the VR-VIII site relative to the parental sequence at the VR-VIII site.

[0146] In some embodiments, the parental sequence is the AAV9 VP1 parental sequence (SEQ ID NO: 30). In some embodiments, the parental sequence is the AAV5 VP1 parental sequence (SEQ ID NO: 37). In some embodiments, the parental sequence is the AAVrh.10 VP1 parental sequence (SEQ ID NO: 109). In some embodiments, the parental sequence is the AAVrh.74 VP1 parental sequence (SEQ ID NO: 119). In some embodiments, the variant polypeptide sequence occurs only at the VR-VIII site. In some embodiments, the engineered AAV9 capsid protein comprises, relative to reference SEQ ID NO: 30, one, two, three, four, five, or more substitutions at positions from 584 to 590 in the VR-VIII site, or one, two, three, four, five, or more substitutions at positions from 585 to 590 in the VR-VIII site. Substitution motifs

[0147] In some embodiments, the engineered capsid proteins described herein comprise a variant polypeptide comprising one or more amino acid substitutions relative to a wild-type or parental capsid protein described herein (referred to herein as a “substitution motif”). In some embodiments, the substitution motif does not comprise amino acid insertions.

[0148] In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at one, two, three, four, five, six, seven, eight, nine, or ten positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at two positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at three positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at four positions relative to a wild-type AAV capsid protein sequence. In some embodiments, theAttorney Docket No. TENA-054 / 01WO 334682-2486 engineered capsid protein comprises a substitution motif comprising an amino acid substitution at five positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at six positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at seven positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at eight positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at one, two, three, four, five, six, seven, or eight of the following positions relative to a wild-type AAV9 capsid protein sequence.

[0149] In some embodiments, the engineered capsid protein comprises a variant polypeptide in the VR-VIII site. In some embodiments, the engineered capsid protein comprises a variant polypeptide at positions 581-595 of the capsid protein, wherein the amino acid numbering is according to the AAV9 VP1 sequence of SEQ ID NO: 30. A person of skill in the art will recognize the equivalent positions in other AAV serotype capsid proteins, e.g., positions 570-583 of the AAV5 VP1 capsid sequence of SEQ ID NO: 37, positions 583-596 of the AAVrh.10 VP1 capsid sequence of SEQ ID NO: 109, and positions 583-596 of the AAVrh.74 VP1 capsid sequence of SEQ ID NO: 119. The variant polypeptide may comprise substitutions across the entire VR-VIII site (e.g., between positions 581-595) or may comprise substitutions in a portion of the VR-VIII site (e.g., between positions 582-591).

[0150] In some embodiments, the engineered capsid protein comprises the variant amino acid sequence of X1X2X3X4X5K at a specified site. In some embodiments, the engineered capsid protein comprises a variant amino acid sequence in the VR-II site. In some embodiments, the engineered capsid protein comprises the variant amino acid sequence X1X2X3X4X5K in a VR-II site of an AAV VP capsid polypeptide sequence. In some embodiments, the engineered capsid protein comprises the variant amino acid sequence X1X2X3X4X5K at amino acid positions 327 to 332 according to the amino acid numbering of SEQ ID NO: 30 (WT AAV9 VP1 protein).Attorney Docket No. TENA-054 / 01WO 334682-2486

[0151] In some embodiments, the site is between amino acids 316 and 333 according to an AAV VP1 amino acid sequence. In some embodiments, the site is at positions 327 to 332, wherein the amino acid numbering is according to SEQ ID NO: 30. In some embodiments, the site is at positions 328 to 333, wherein the amino acid numbering is according to SEQ ID NO: 109. In some embodiments, the site is at positions 328 to 333, wherein the amino acid numbering is according to SEQ ID NO: 119.

[0152] In some embodiments, the variant amino acid sequence X1X2X3X4X5K is QTDGVK (SEQ ID NO: 123) In some embodiments, the variant amino acid sequence X1X2X3X4X5K is QQDGTK (SEQ ID NO: 124).

[0153] In some embodiments, the engineered capsid protein comprises a variant amino acid sequence within the last 35 amino acid positions of the capsid protein. In some embodiments, the engineered capsid protein comprises the variant amino acid sequence X1X2X3GX4within the last 35 amino acid positions of an AAV VP capsid polypeptide sequence. In some embodiments, the engineered capsid protein comprises the variant amino acid sequence X1X2X3GX4 at amino acid positions 716 to 720 according to the amino acid numbering of SEQ ID NO: 30.

[0154] In some embodiments, the capsid protein comprises the variant amino acid sequence of X1X2X3GX4 at a specified site. In some embodiments, the site is at amino acid positions 716 to 720 wherein the amino acid numbering is according to SEQ ID NO: 30. In some embodiments, the site is at positions 718 to 722, wherein the amino acid numbering is according to SEQ ID NO: 37. In some embodiments, the site is at positions 718 to 722, wherein the amino acid numbering is according to SEQ ID NO: 109. In some embodiments, the site is at positions 704 to 708, wherein the amino acid numbering is according to SEQ ID NO: 119.

[0155] In some embodiments, the variant amino acid sequence X1X2X3GX4 is selected from the group consisting of: NQYGV (SEQ ID NO: 125), NVHGV (SEQ ID NO: 126), NTHGV (SEQ ID NO: 127), and NTRGE (SEQ ID NO: 128). In some embodiments, the variant amino acid sequence X1X2X3GX4 is NQYGV (SEQ ID NO: 125). In some embodiments, the variant amino acid sequence X1X2X3GX4is NTRGE (SEQ ID NO: 128).

[0156] In some embodiments, the variant polypeptide comprises one or more substitutions (e.g., a substitution motif). In some embodiments, the non-naturallyAttorney Docket No. TENA-054 / 01WO 334682-2486 occurring amino acid motif comprises a substitution motif and does not comprise an insertion motif

[0157] In some embodiments, the engineered AAV9 capsid protein comprises an amino acid sequence of X1DVQX2X3PGFX4X5X6X7X8 (SEQ ID NO: 44) at the VR-VIII site (e.g., of a wild-type AAV9 capsid protein or a variant thereof), wherein each of X1, X2, X3, X4, X5, X6, X7, and X8is any amino acid.

[0158] In some embodiments, X1is alanine (A).

[0159] In some embodiments, X2 is glutamine (Q).

[0160] In some embodiments, X7 is threonine (T).

[0161] In some embodiments, X5is alanine (A) or proline (P).

[0162] In some embodiments, X6 is glutamine (Q) or glutamic acid (E).

[0163] In some embodiments, X4 is glutamine (Q), glycine (G), arginine (R), asparagine (N), histidine (H), methionine (M), proline (P), or serine (S).

[0164] In some embodiments, X8 is glutamic acid (E), methionine (M), glutamine (Q), aspartic acid (D), leucine (L), alanine (A), cysteine (C), histidine (H), phenylalanine (F), tyrosine (Y), threonine (T), valine (V), isoleucine (I), serine (S), or asparagine (N). In some embodiments, X8is glutamic acid (E).

[0165] In some embodiments, X3 is leucine (L), histidine (H), valine (V), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), methionine (M), phenylalanine (F), proline (P), threonine (T), or tyrosine (Y).

[0166] In some embodiments, X1 is A, X2 is Q, X7 is T, and / or the capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4X5X6TX8 (SEQ ID NO: 45), wherein each of X3, X4, X5, X6, and X8is any amino acid.

[0167] In some embodiments, X5 is A or P, and X6 is Q or E, and / or the capsid protein comprises in the VR-VIII site an amino acid sequence of X1DVQX2X3PGFX4AQX7X8(SEQ ID NO: 46), X1DVQX2X3PGFX4AEX7X8(SEQ ID NO: 47), X1DVQX2X3PGFX4PQX7X8(SEQ ID NO: 48), X1DVQX2X3PGFX4PEX7X8(SEQ ID NO: 49), ADVQQX3PGFX4AQTX8 (SEQ ID NO: 50), ADVQQX3PGFX4AETX8 (SEQ ID NO: 52), ADVQQX3PGFX4PQTX8(SEQ ID NO: 53), or ADVQQX3PGFX4PETX8(SEQ ID NO: 53), wherein each of X1, X2, X3, X4, X7, and X8is any amino acid.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0168] In some embodiments, X3 is selected from L, H, V, C, Q, G, I, M, F, P, T, or Y, X4 is selected from Q, G, R, N, H, M, P, or S, and X8 is selected from E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N. In some embodiments, X3is L and X8is E.

[0169] In some embodiments, X4 is Q, X5 is A, and X6 is Q, and / or the capsid protein comprises in the VR-VIII site an amino acid sequence of X1DVQX2X3PGFQAQX7X8 (SEQ ID NO: 54) or ADVQQX3PGFQAQTX8(SEQ ID NO: 55), wherein each of X1, X2, X3, X7, and X8is any amino acid.

[0170] In some embodiments, X3 is L, and X8 is E, and / or the capsid protein comprises in the VR-VIII site an amino acid sequence of X1DVQX2LPGFX4X5X6X7E (SEQ ID NO: 56) or ADVQQLPGFX4X5X6TE (SEQ ID NO: 57), wherein each of X1, X2, X4, X5, X6, and X7 is any amino acid. In some embodiments, X4 is Q.

[0171] In some embodiments, X4 is Q, and / or the capsid protein comprises in the VR-VIII site an amino acid sequence of X1DVQX2X3PGFQX5X6X7X8(SEQ ID NO: 58) or ADVQQX3PGFQX5X6TX8 (SEQ ID NO: 59), wherein each of X1, X2, X3, X5, X6, X7, and X8 is any amino acid.

[0172] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of X1DVQX2X3PGFX4AX6X7X8,wherein each of X1, X2, X3, X4, X6, X7, and X8 is any amino acid (SEQ ID NO: 129).

[0173] In some embodiments, the engineered AAV capsid comprises in the VR-VIII site an amino acid sequence of X1DVQX2X3PGFX4X5QX7X8,wherein each of X1, X2, X3, X4, X5, X7, and X8 is any amino acid (SEQ ID NO: 130).

[0174] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4PETX8,wherein each of X3, X4and / or X8 is any amino acid (SEQ ID NO: 131). In some embodiments, X3 is L, H, S, V, C, Q, G, I, M, F, P, T, or Y. In some embodiments, X4 is Q, G, R, N, H, M, P, or S. In some embodiments, X8is E, G, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.

[0175] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4AQTX8, wherein each of X3, X4 and / or X8is any amino acid (SEQ ID NO: 132). In some embodiments, X3is L, H, S, V, C, Q, G, I, M, F, P, T, or Y. In some embodiments, X4is Q, G, R, N, H, M, P, or S. In some embodiments, X8 is E, G, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0176] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFQAQTE, wherein X3 is any amino acid (SEQ ID NO: 133).

[0177] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4X5X6X7X8, wherein each of X3, X4, X5, X6, X7, and X8is any amino acid (SEQ ID NO: 134).

[0178] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4X5X6X7E, wherein each of, X3, X4, X5, X6, and X7 is any amino acid (SEQ ID NO: 135).

[0179] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQHPGFX4X5X6TE, wherein each of X4, X5, and X6 is any amino acid (SEQ ID NO: 136).

[0180] In some embodiments, the engineered AAV9 capsid protein comprises, consists essentially of, or consists of a sequence having at least about 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%) identity to any one of the following sequences at the VR-VIII site (positions 581-594 relative to reference sequence SEQ ID NO: 30), with up to 1, 2, or 3 substitutions: ADVQQLPGFQAQTEW (SEQ ID NO: 60), ADVQQHPGFQAQTEW (SEQ ID NO: 61), ADVQQVPGFQAQTMW (SEQ ID NO: 62), ADVQQVPGFQAQTQW (SEQ ID NO: 63), ADVQQLPGFGAQTEW (SEQ ID NO: 64), ADVQQLPGFRPETEW (SEQ ID NO: 65), and ADVQQLPGFNAQTEW (SEQ ID NO: 66).

[0181] In some embodiments, the engineered AAV9 capsid protein comprises in the VR-VIII site an amino acid sequence selected from ADVQQLPGFQAQTEW (SEQ ID NO: 60), ADVQQHPGFQAQTEW (SEQ ID NO: 61), ADVQQVPGFQAQTMW (SEQ ID NO: 62), ADVQQVPGFQAQTQW (SEQ ID NO: 63), ADVQQLPGFGAQTEW (SEQ ID NO: 64), ADVQQLPGFRPETEW (SEQ ID NO: 65), and ADVQQLPGFNAQTEW (SEQ ID NO: 66).

[0182] In some embodiments, the engineered AAV9 capsid protein comprises in the VR-VIII site an amino acid sequence selected from ADVQQLPGFQAQTEW (SEQ ID NO: 60) and ADVQQHPGFQAQTEW (SEQ ID NO: 61).Attorney Docket No. TENA-054 / 01WO 334682-2486

[0183] In some embodiments, the engineered AAV9 capsid protein comprises one or more amino acid substitutions selected from the group consisting of A581R, A581N, A581D, A581C, A581Q, A581E, A581G, A581H, A581I, A581L, A581K, A581M, A581F, A581P, A581O, A581S, A581T, A581W, A581Y, A581V, T582D, N583V, H584Q, Q585T, Q585C, Q585V, Q585L, Q585N, Q585S, Q585P, Q585A, Q585M, Q585E, Q585Y, Q585G, Q585H, Q585I, Q585R, Q585D, Q585K, Q585F, Q585O, Q585, S586D, S586T, S586G, S586K, S586M, S586N, S586I, S586Q, S586L, S586P, S586F, S586R, S586A, S586C, S586E, S586H, S586O, S586W, S586Y, S586V, A587P, A587S, A587N, Q588G, Q588R, Q588V, A589F, A589T, Q590I, Q590S, Q590N, Q590G, Q590D, Q590R, Q590H, Q590T, Q590M, Q590F, Q590Y, Q590L, Q590A, Q590C, Q590E, Q590K, Q590P, Q590O, Q590W, Q590V, A591I, A591R, A591N, A591D, A591C, A591Q, A591E, A591G, A591H, A591 L, A591K, A591M, A591F, A591P, A591O, A591S, A591T, A591W, A591Y, A591V, Q592I, Q592R, Q592N, Q592D, Q592C, Q592A, Q592E, Q592G, Q592H, Q592 L, Q592K, Q592M, Q592F, Q592P, Q592O, Q592S, Q592T, Q592W, Q592Y, Q592V, T593I, T593R, T593N, T593D, T593C, T593A, T593E, T593G, T593H, T593L, T593K, T593M, T593F, T593P, T593O, T593S, T593Q, T593W, T593Y, T593V, G594I, G594R, G594N, G594D, G594C, G594A, G594E, G594Q, G594H, G594 L, G594K, G594M, G594F, G594P, G594O, G594S, G594T, G594W, G594Y, and G594V.

[0184] In some embodiments, the engineered capsid protein may comprise one or more (e.g., three, four, five, six, seven or eight) amino acid substitutions selected from the group consisting of S586L, T582D, N583V, H584Q, A587P, Q588G, A589F, and G594E, relative to reference sequence SEQ ID NO: 30.

[0185] In some embodiments, the engineered capsid protein may comprise one or more (e.g., three, four, five, six, seven or eight) amino acid substitutions selected from the group consisting of S586H, T582D, N583V, H584Q, A587P, Q588G, A589F, and G594E, relative to reference sequence SEQ ID NO: 30.

[0186] In some embodiments, the engineered capsid protein comprises one or more amino acid substitutions selected from the group consisting of: D327Q, D327V, N328T, N328Q, N328S, N329D, G330N, and V331T. In some embodiments, engineered the capsid protein comprises the amino acid substitutions D327Q, N328T, and N329D. InAttorney Docket No. TENA-054 / 01WO 334682-2486 some embodiments, the engineered capsid protein comprises the amino acid substitutions D327Q, N328Q, N329D, V331T.

[0187] In some embodiments, the engineered capsid protein comprises one or more amino acid substitutions selected from the group consisting of: N716D, T717Q, T717M, T717V, T717A, T717P, T717I, E718Y, E718R, E718H, E718N, E718M, E718Q, V720E, V720Q, V720I, and V720A. In some embodiments, the capsid protein comprises the amino acid substitutions T717Q and E718Y. In some embodiments, the capsid protein comprises the amino acid substitutions E718R and V720E.

[0188] In some embodiments, the engineered AAV9 capsid protein comprises any substitution and / or insertion motif described herein. In some embodiments, the engineered capsid protein comprises a substitution motif having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any substitution motif described herein. In some embodiments, the engineered capsid protein comprises an insertion motif having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any insertion motif described herein.

[0189] It should be noted that the above modified VR-VIII motifs are described in the context of AAV9 capsid proteins for illustrative purposes only and are not meant to be limited to AAV9 capsid proteins. Instead, any modified VR-VIII motif described herein can be applied to other AAV capsid proteins of a different serotype (e.g., AAV5, AAVrh.10, or AAVrh.74), for example, by replacing the wild-type sequence at the VR-VIII site of the corresponding capsid protein (e.g., amino acid positions 570 to 583 of wild- type AAV5 VP1 capsid protein sequence according to SEQ ID NO: 58, amino acid positions 583 to 596 of wild-type AAVrh.10 VP1 capsid protein sequence, or amino acid positions 583 to 596 of wild-type AAVrh.74 VP1 capsid protein sequence) with any of the modified VR-VIII motifs described herein to generate a variant of the capsid protein of a particular serotype. In some embodiments, the engineered capsid protein is a variant of an AAV5, AAV9, AAVth.10, or AAVrh.74 capsid protein.

[0190] In some embodiments, the engineered AAV9 capsid protein comprises one, two, three, four, five, or more insertions in the VR-VIII site. In some embodiments, theAttorney Docket No. TENA-054 / 01WO 334682-2486 engineered AAV9 capsid protein comprises, relative to reference SEQ ID NO: 30, one, two, three, four, five, or more insertions at positions from 584 to 590 in the VR-VIII site, or one, two, three, four, five, or more insertions at positions from 585 to 590 in the VR- VIII site.

[0191] In some embodiments, the engineered capsid protein comprises an insertion polypeptide or insertion motif compared to the wild-type or parental capsid protein. In some embodiments, the engineered capsid protein additionally comprises one or more amino acid substitutions in the amino acid sequence of the wild-type or parental capsid protein sequence from which it is derived. In some embodiments, the insertion motif is inserted at a surface loop region of the capsid protein, for example, at a VR-IV, VR-V, VR-VII and / or VR-VIII site, as described.

[0192] In some embodiments, the insertion motif comprises or consists of the amino acid sequence RGDXKGL, wherein X can be any amino acid. In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDAARL” (SEQ ID NO: 67); and / or the engineered capsid protein comprises an amino acid sequence of “RGDAARL” (SEQ ID NO: 67).

[0193] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “SHVRGDL” (SEQ ID NO: 68); and / or the engineered capsid protein comprises an amino acid sequence of “SHVRGDL” (SEQ ID NO: 68).

[0194] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “VVSSGAR” (SEQ ID NO: 69); and / or the engineered capsid protein comprises an amino acid sequence of “VVSSGAR” (SEQ ID NO: 69).

[0195] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “VRGD” (SEQ ID NO: 70); and / or the engineered capsid protein comprises an amino acid sequence of “VRGD” (SEQ ID NO: 70).

[0196] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RTDLKGL” (SEQ ID NO: 71); and / or the engineered capsid protein comprises an amino acid sequence of “RTDLKGL” (SEQ ID NO: 71).

[0197] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDTKGL” (SEQ ID NO: 138); and / or the engineered capsid protein comprises an amino acid sequence of “RGDTKGL” (SEQ ID NO: 138).Attorney Docket No. TENA-054 / 01WO 334682-2486

[0198] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDAKGL” (SEQ ID NO: 139); and / or the engineered capsid protein comprises an amino acid sequence of “RGDAKGL” (SEQ ID NO: 139).

[0199] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDVKGL” (SEQ ID NO: 140); and / or the engineered capsid protein comprises an amino acid sequence of “RGDVKGL” (SEQ ID NO: 140).

[0200] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDLVST” (SEQ ID NO: 187); and / or the engineered capsid protein comprises an amino acid sequence of “RGDLVST” (SEQ ID NO: 187).

[0201] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDGGVL” (SEQ ID NO: 188); and / or the engineered capsid protein comprises an amino acid sequence of “RGDGGVL” (SEQ ID NO: 188).

[0202] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDHASW” (SEQ ID NO: 189); and / or the engineered capsid protein comprises an amino acid sequence of “RGDHASW” (SEQ ID NO: 189).

[0203] The insertion motif can occur (e.g., be inserted) at any position of the capsid protein, for example, at a surface or an exposed region of the capsid protein. In some embodiments, the engineered AAV capsid protein comprises an insertion motif as described herein inserted at a surface loop region of the capsid protein, e.g., the VR-I, VR-II, VR-IV, VR-V, VR-VII, and / or VR-VIII site of the capsid protein. In some embodiments, the engineered capsid protein comprises an insertion motif as described inserted at the VR-IV and / or the VR-VIII site of the capsid protein. In certain of these embodiments, the engineered capsid protein additionally comprises one or more amino acid substitutions in the same VR site as the insertion or at a different location from the insertion.

[0204] In some embodiments, the engineered capsid protein is an engineered AAV9 capsid protein that comprises an insertion polypeptide or insertion motif at the VR-VIII site, e.g., between amino acids 588 (glutamine (Q)) and 589 (alanine (A)) within the VR- VIII site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 30. In some embodiments, the engineered AAV9 capsid protein further comprises one or more amino acid substitutions within the VR-VIII site, including, for example, at one orAttorney Docket No. TENA-054 / 01WO 334682-2486 more of amino acid positions 587-590 in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 30. In certain of these embodiments, the insertion motif comprises an amino acid sequence of RGDAARL (SEQ ID NO: 67), RTDLKGL (SEQ ID NO: 71), YPSTGSG (SEQ ID NO: 72), FAGSLTRA (SEQ ID NO: 73), DRTLTTR (SEQ ID NO: 74), RIAGRDV (SEQ ID NO: 75), or SLGSGVR (SEQ ID NO: 76), RGDTKGL (SEQ ID NO: 138), RGDAKGL (SEQ ID NO: 139), RGDVKGL (SEQ ID NO: 140), RGDLVST (SEQ ID NO: 187), RGDGGVL (SEQ ID NO: 188), or RGDHASW” (SEQ ID NO: 189).

[0205] In some embodiments, provided herein is an engineered capsid protein comprising an amino acid sequence “ATNHQSX1X2X3X4AQTGW” (SEQ ID NO: 150) in the VR-VIII site, wherein X1, X2, X3, and X4 can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (for example, between amino acid positions 588 and 589 of wild-type AAV9 VP1 capsid protein sequence). In some embodiments, provided herein is an engineered capsid protein comprising an amino acid sequence “ATNHQSX1X2X3X4AQTEW” (SEQ ID NO: 151) wherein X1, X2, X3, and X4 can individually be any amino acid, and an insertion motif is inserted between X2and X3(for example, between amino acid positions 588 and 589 of wild-type AAV9 VP1 capsid protein sequence). In some embodiments, provided herein is an engineered capsid protein comprising an amino acid sequence “ATNHQLX1X2X3X4AQTGW” (SEQ ID NO: 152) in the VR-VIII site, wherein X1, X2, X3, and X4can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (for example, between amino acid positions 588 and 589 of wild-type AAV9 VP1 capsid protein sequence). In some embodiments, provided herein is an engineered capsid protein comprising an amino acid sequence “ATNHQLX1X2X3X4AQTEW” (SEQ ID NO: 153) in the VR-VIII site, wherein X1, X2, X3, and X4 can individually be any amino acid, and an insertion motif is inserted between X2and X3(for example, between amino acid positions 588 and 589 of wild-type AAV9 VP1 capsid protein sequence).

[0206] In some embodiments, the engineered capsid protein is an engineered AAV9 capsid protein that comprises an insertion polypeptide or insertion motif at the VR-IV site, e.g., between amino acids 453 (glycine (G)) and 454 (serine (S)), and / or between amino acids 456 (glutamine (Q)) and 457 (asparagine (N)), within the VR-IV site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 30. In certain of these embodiments, the insertion motif comprises an amino acid sequence of SHVRGDL (SEQAttorney Docket No. TENA-054 / 01WO 334682-2486 ID NO: 68), VVSSGAR (SEQ ID NO: 69), PQYGRGG (SEQ ID NO: 77), LQVSRVS (SEQ ID NO: 78), VRSYSSN (SEQ ID NO: 79), “TMRVGSL” (SEQ ID NO: 80), GAYSRGV (SEQ ID NO: 81), LRGGSLG (SEQ ID NO: 82), or “VYGTGVR” (SEQ ID NO: 83).

[0207] In some embodiments, the engineered capsid protein is an engineered AAV5 capsid protein that comprises an insertion polypeptide or insertion motif at the VR-VIII site, e.g., between amino acids 574 (glutamine (Q)) and 575 (serine (S)) within the VR- VIII site in reference to the wild-type full-length AAV5 capsid protein of SEQ ID NO: 37. In certain of these embodiments, the insertion motif comprises an amino acid sequence of DKLIIVS (SEQ ID NO: 84), AEDRTKL (SEQ ID NO: 85), LSASASL (SEQ ID NO: 86), LADQTKL (SEQ ID NO: 87), LLLKLQE (SEQ ID NO: 88), ELPVKTG (SEQ ID NO: 89), LDLKVVG (SEQ ID NO: 90), or RDAVL (SEQ ID NO: 91).

[0208] In some embodiments, the engineered AAV9 capsid protein comprises an insertion peptide sequence or insertion motif at the VR-VIII site, e.g., between amino acids 588 (glutamine (Q)) and 589 (alanine (A)) within the VR-VIII site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 30. The insertion motif can be any motif as described herein, including those provided in Table 3B below. In some embodiments, the engineered AAV9 capsid protein further comprises one or more amino acid substitutions within the VR-VIII site, including, for example, at one or more of amino acid positions 587-590 in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 30.

[0209] In some embodiments, the engineered AAV9 capsid protein further comprises one or more amino acid substitutions within the VR-VIII site, including, for example, at one or more of amino acid positions 586 and / or 594 in reference to the wild- type full-length AAV9 capsid protein of SEQ ID NO: 30. In certain of these embodiments, the engineered AAV9 capsid protein comprises a sequence of “ATNHQSX1X2X3X4AQTEW” (SEQ ID NO: 151) at the VR-VIII site, where X1, X2, X3, and X4can individually be any amino acid, and an insertion motif is inserted between X2and X3 (i.e., between amino acid positions 588 and 589). In certain of these embodiments, the engineered AAV9 capsid protein comprises a sequence of “ATNHQLX1X2X3X4AQTGW” (SEQ ID NO: 152) at the VR-VIII site, where X1, X2, X3, and X4 can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (i.e., between amino acid positions 588 and 589). In certain of these embodiments,Attorney Docket No. TENA-054 / 01WO 334682-2486 the engineered AAV9 capsid protein comprises a sequence of “ATNHQLX1X2X3X4AQTEW” (SEQ ID NO: 153) at the VR-VIII site, where X1, X2, X3, and X4can individually be any amino acid, and an insertion motif is inserted between X2and X3 (i.e., between amino acid positions 588 and 589). Table 3B. Exemplary engineered AAV9 capsid protein VR-VIII sequences with superior performance in nonhuman primates.

[0210] In some embodiments, the engineered AAV9 capsid protein comprises a “substitution + insertion motif”, wherein the substitution + insertion motif comprises: an insertion peptide sequence or insertion motif at the VR-VIII site, e.g., between aminoAttorney Docket No. TENA-054 / 01WO 334682-2486 acids 588 (glutamine (Q)) and 589 (alanine (A)) within the VR-VIII site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 30, and (ii) one or more amino acid substitutions within the VR-VIII site, including, for example, at one or more of amino acid positions 586, 587, 588, 590, and 594 in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 30. In some embodiments, the substitution + insertion motif comprises amino acids 586 to 594, including the insertion motif, within the VR-VIII site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 30. In some embodiments, the substitution + insertion motif can be any disclosed herein, including those provided in Table 3C below. Table 3C. Exemplary substitution + insertion motifsAttorney Docket No. TENA-054 / 01WO 334682-2486

[0211] In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 67, 71, 138-140, and 187-189. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 138. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 139. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 187. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 188. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 189.

[0212] In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 67, 71, 138-140, and 187-189, with up to 1, 2, or 3 amino acid substitutions. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 138, 139, and 187-189, with up to 1, 2, or 3 amino acid substitutions.

[0213] In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises, consists essentially of, or consists of an amino acid sequence that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 141, or the amino acid sequence set forth SEQ ID NO: 141. In some embodiments, the engineered AAV9 capsid protein, at the VR- VIII site, comprises, consists essentially of, or consists of an amino acid sequence that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 143, or the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises, consists essentially of, or consists of an amino acid sequenceAttorney Docket No. TENA-054 / 01WO 334682-2486 that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NOs: 190, or the amino acid sequence set forth in SEQ ID NO: 190. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises, consists essentially of, or consists of an amino acid sequence that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 191, or the amino acid sequence set forth in SEQ ID NOs: 191. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises, consists essentially of, or consists of an amino acid sequence that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 192, or the amino acid sequence set forth in SEQ ID NO: 192.

[0214] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 32, and comprises the amino acid sequence of ATNHQSSVRGDTKGLAGAQTGW (SEQ ID NO: 141) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 30.

[0215] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 32, and comprises the amino acid sequence of ATNHQSSVRTDLKGLAGAQTGW (SEQ ID NO: 143) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 30.

[0216] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 32, and comprises the amino acid sequence of ATNHQENRRGDLVSTTQAQTGW (SEQ ID NO: 190) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 30.

[0217] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 32, and comprises the amino acid sequence of ATNHQENRRGDGGVLAQAQTGW (SEQ ID NO: 191) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 30.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0218] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 32, and comprises the amino acid sequence of ATNHQSSVRGDHASWAQAQTGW (SEQ ID NO: 192) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 30.

[0219] Exemplary engineered capsid protein sequences are provided in Table 3D below. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence set forth in any one of SEQ ID NOs: 92- 102 and 173-186, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 92-102 and 173-186.

[0220] In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 92 or SEQ ID NO: 93. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 92. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 93.

[0221] In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 173 or 175. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 173. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 175.

[0222] In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 183- 186. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 183. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 184. In some embodiments, the engineered capsidAttorney Docket No. TENA-054 / 01WO 334682-2486 protein comprises, consists of, or consists essentially of SEQ ID NO: 185. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 186.

[0223] In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 199-201. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 199. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 200. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 201. Table 3D. Exemplary engineered AAV capsid protein sequencesAttorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486Attorney Docket No. TENA-054 / 01WO 334682-2486

[0224] In some embodiments, the rAAV virus or virion comprises a wild-type AAVrh.10 capsid protein or a variant thereof as known in the art. In some embodiments, the rAAV virus or virion comprises a wild-type AAVrh.74 capsid protein or a variant thereof as known in the art. In some embodiments, the rAAV virus or virion comprises an AAV-SLB101 capsid protein or a variant thereof as known in the art or described in, e.g., WO 2021 / 072197, which is incorporated by reference herein in its entirety. In someAttorney Docket No. TENA-054 / 01WO 334682-2486 embodiments, the rAAV virus or virion comprises an AAVmod capsid protein or a variant thereof as known in the art or described in, e.g., WO 2022 / 173847 or in Olivieri et al. (2021) 24th Annual Meeting of the American Society of Gene & Cell Therapy available at https: / / www.affiniatx.com / pdf / asgct_2021_olivieri.pdf, both of which are incorporated by reference herein in their entireties. In some embodiments, the rAAV virus or virion comprises an AAVmut1dec1, AAVdeco1, and / or AAVmut1 capsid protein or a variant thereof as known in the art or described in, e.g., WO 2022 / 173847, which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises an AAVcc.47 capsid protein or a variant thereof as known in the art or described in, e.g., Gonzalez et al. Nature Communications 13:5947 (2022), which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises an AAVHSC16 capsid protein or a variant thereof as known in the art or described in, e.g., Smith et al. Molecular Therapy Methods & Clinical Development 26:224-238 (2022), which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises a MyoAAV capsid protein or variant thereof as known in the art or described in, e.g., Tabebordbar et al. Cell 184(19):4919- 4938. (2021), which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises a MyoAAV-4E, MyoAAV-3F, MyoAAV- 4A, or MyoAAV-4D capsid protein or variant thereof as known in the art or described in, e.g., Tabebordbar et al, which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises a 4D-C102 or C102 capsid protein or a variant thereof as known in the art or described in, e.g., US2021 / 0380643, which is incorporated by reference herein in its entirety.

[0225] In some embodiments, the rAAV virus or virion comprises a capsid protein (such as any described herein) and a vector genome, and the vector genome comprises an expression cassette flanked by ITRs. In some embodiments, the rAAV virus or virion specifically transduces heart cells and / or cardiomyocytes. In some embodiments, the rAAV virus or virion traffics to the heart. In some embodiments, the rAAV virus or virion traffics to at least one organ other than the liver.

[0226] In some embodiments, the rAAV virus or virion exhibits a higher transduction efficiency (e.g., a higher heart transduction efficiency) than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 30. In some embodiments, the rAAV virus or virion exhibits a higher transduction efficiency (e.g., a higher heartAttorney Docket No. TENA-054 / 01WO 334682-2486 transduction efficiency) in a primate or as assessed in a primate than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 30.

[0227] In some embodiments, administration of the rAAV virus or virion to a subject leads to a lower liver viral load than administration of an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 30. In some embodiments, administration of the rAAV virus or virion to a subject leads to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times lower liver viral load than administration of an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 30. In some embodiments, administration of the rAAV virus or virion to a subject leads to a lower liver viral load in a primate or as assessed in a primate than administration of an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 30. In some embodiments, administration of the rAAV virus or virion to a subject leads to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times lower liver viral load in a primate or as assessed in a primate than administration of an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 30.

[0228] In some embodiments, the rAAV virus or virion exhibits a higher heart-to- liver transduction ratio than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 30. In some embodiments, the rAAV virion exhibits a heart-to- liver transduction ratio which is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 30. In some embodiments, the rAAV virus or virion exhibits a higher heart-to-liver transduction ratio in a primate or as assessed in a primate than an rAAV virus or virion having a wild- type AAV9 VP1 capsid protein of SEQ ID NO: 30. In some embodiments, the rAAV virion exhibits a heart-to-liver transduction ratio which is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher in a primate or as assessed in a primate than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 30.

[0229] In some embodiments, the rAAV virus or virion is replication defective, in that the rAAV virus or virion cannot independently further replicate and package its genome. For example, when a cardiac cell is targeted with rAAV virions, the transgene is expressed in the targeted cardiac cell, however, due to the fact that the targeted cardiac cell lacks AAV rep and cap genes and accessory function genes, the rAAV is not able to replicate.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0230] In some embodiments, rAAV virus or virion encapsulating the expression cassettes as described herein can be produced using helper-free production. rAAVs are replication-deficient viruses and normally require components from a live helper virus, such as adenovirus, in a host cell for packaging of infectious rAAV virions. rAAV helper- free production systems allow the production of infectious rAAV virions without the use of a live helper virus. In the helper-free system, a host packaging cell line is co- transfected with three plasmids. A first plasmid may contain adenovirus gene products (e.g., E2A, E4, and VA RNA genes) needed for the packaging of rAAV virions. A second plasmid may contain required AAV genes (e.g., REP and CAP genes). A third plasmid contains the polynucleotide sequence encoding the transgene of interest and a promoter flanked by ITRs. A host packaging cell line can be, for example, AAV-293 host cells. Suitable host cells contain additional components required for packaging infectious rAAV virions that are not supplied by the plasmids. In some embodiments, the CAP genes can encode, for example, AAV capsid proteins as described herein. Pharmaceutical compositions

[0231] In some embodiments, the expression cassette and / or vector comprising a polynucleotide encoding a SYNPO2LA protein for use in the present technology is in a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, or excipients for parenteral delivery. Pharmaceutically acceptable carriers, diluents, or excipients can include vehicles that are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. Illustrative pharmaceutical forms suitable for injectable use include, e.g., sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.

[0232] In some embodiments, the pharmaceutical composition comprises about 1×108genome copies per milliliter (GC / mL), about 5×108GC / mL, about 1×109GC / mL,Attorney Docket No. TENA-054 / 01WO 334682-2486 about 5×109GC / mL, about 1×1010GC / mL, about 5×1010GC / mL, about 1×1011GC / mL, about 5×1011GC / mL, about 1×1012GC / mL, about 5×1012GC / mL, about 5×1013GC / mL, about 1×1014GC / mL, or about 5×1014GC / mL of the vector (e.g., rAAV virus or virion).

[0233] In some embodiments, the pharmaceutical composition comprises about 1×108viral genomes per milliliter (vg / mL), about 5×108vg / mL, about 1×109vg / mL, about 5×109vg / mL, about 1×1010vg / mL, about 5×1010vg / mL, about 1×1011vg / mL, about 5×1011vg / mL, about 1×1012vg / mL, about 5×1012vg / mL, about 5×1013vg / mL, about 1×1014vg / mL, or about 5×1014vg / mL of the vector (e.g., rAAV virus or virion).

[0234] In some embodiments, the pharmaceutical composition comprises less than about 1×1015viral genomes per milliliter (vg / mL), less than about 5×1014vg / mL, less than about 1×1014vg / mL, less than about 5×1013vg / mL, less than about 1×1013vg / mL, less than about 5×1012vg / mL, less than about 1×1012vg / mL, less than about 5×1011vg / mL, or less than about 1×1011vg / mL of the vector (e.g., rAAV virus or virion).

[0235] In some embodiments, the pharmaceutical composition comprises less than about 1×1014viral genomes per milliliter (vg / mL) or less than about 1×1013vg / mL of the vector (e.g., rAAV virus or virion).

[0236] In some embodiments, the pharmaceutical composition comprises from about 1×1011viral genomes per milliliter (vg / mL) to about 1×1015vg / mL of the vector (e.g., rAAV virus or virion). In some embodiments, the pharmaceutical composition comprises from about 1×1011viral genomes per milliliter (vg / mL) to about 1×1014vg / mL of the vector (e.g., rAAV virus or virion). In some embodiments, the pharmaceutical composition comprises from about 1×1012viral genomes per milliliter (vg / mL) to about 1×1014vg / mL of the vector (e.g., rAAV virus or virion). In some embodiments, the pharmaceutical composition comprises from about 1×1012viral genomes per milliliter (vg / mL) to about 1×1013vg / mL of the vector (e.g., rAAV virus or virion). In some embodiments, the pharmaceutical composition comprises from about 1×1012viral genomes per milliliter (vg / mL) to about 6×1013vg / mL of the vector (e.g., rAAV virus or virion).

[0237] In some embodiments, the pharmaceutical composition comprises any amount or concentration range of the vector (e.g., rAAV virus or virion) between the values referenced herein.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0238] In some embodiments, the pharmaceutical composition is administered in a total volume of about 1 mL, about 5 mL, about 10 mL, about 20 mL, about 25mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, about 50 mL, about 55 mL, about 60 mL, about 65 mL, about 70 mL, about 75 mL, about 80 mL, about 85 mL, about 90 mL, about 95 mL, about 100 mL, about 105 mL, about 110 mL, about 115 mL, about 120 mL, about 125 mL, about 130 mL, about 135 mL, about 140 mL, about 145 mL, about 150 mL, about 155 mL, about 160 mL, about 165 mL, about 170 mL, about 175 mL, about 180 mL, about 185 mL, about 190 mL, about 200 mL, about 205 mL, about 210 mL, about 215 mL, or about 220 mL.

[0239] In some embodiments, the pharmaceutical composition can be formulated (e.g., injectable, lyophilized, liquid formulations, or oral formulations) to be compatible with its intended route of administration. Examples of routes of administration include oral administration, extracorporeal administration, parenteral administration, intravenous administration, subcutaneous administration, or local administration to the heart. In some embodiments, local administration is by direct injection into the heart or cardiac tissue, intracoronary administration, or retrograde coronary sinus infusion. For example, a pharmaceutical composition provided herein can be administered systemically by intravenous administration (e.g., injection or infusion) or locally (e.g., by direct injection into the heart or cardiac tissue, intracoronary administration, or retrograde coronary sinus infusion). Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0240] In some embodiments, the pharmaceutical composition can be co- formulated in the same dosage unit or can be individually formulated in separate dosage units. The term “dosage unit” herein refers to a portion of a pharmaceutical composition that contains an amount of a therapeutic agent suitable for a single administration toAttorney Docket No. TENA-054 / 01WO 334682-2486 provide a therapeutic effect. Such dosage units may be administered one to a plurality (e.g., 1 to about 10, 1 to 8, 1 to 6, 1 to 4, or 1 to 2) of times per day, or as many times as needed to elicit a therapeutic response. Kits

[0241] In some embodiments, provided are kits comprising a container housing a pharmaceutical composition as described herein.

[0242] The kit can include any of compositions described herein, either mixed together or individually packaged, and in dry or hydrated form. The rAAV virions and / or other agents described herein can be packaged separately into discrete vials, bottles or other containers. Alternatively, any of the rAAV virions and / or agents described herein can be packaged together as a single composition, or as two or more compositions that can be used together or separately. The compounds and / or agents described herein can be packaged in appropriate ratios and / or amounts to facilitate conversion of selected cells across differentiation boundaries to form cardiac progenitor cells and / or cardiomyocytes.

[0243] The kit can include instructions for administering those compositions, compounds and / or agents. Such instructions can provide the information described throughout this application. The rAAV virion or pharmaceutical composition can be provided within any of the kits in the form of a delivery device. Alternatively, a delivery device can be separately included in the kits, and the instructions can describe how to assemble the delivery device prior to administration to a subject.

[0244] Any of the kits can also include syringes, catheters, scalpels, sterile containers for sample or cell collection, diluents, pharmaceutically acceptable carriers, and the like. The kits can provide other factors such as any of the supplementary factors or drugs described herein for the compositions in the preceding section or other parts of the application. Cells

[0245] In some embodiments, provided are isolated cells or populations of cells comprising one or more vectors (e.g., rAAV viruses or virions) described herein.

[0246] In some embodiments, the cell is a cardiac cell. As used herein the term “cardiac cell” refers to any cell present in the heart that provides a cardiac function, suchAttorney Docket No. TENA-054 / 01WO 334682-2486 as heart contraction or blood supply, or otherwise serves to maintain the structure of the heart. Cardiac cells as used herein encompass cells that exist in the epicardium, myocardium or endocardium of the heart. Cardiac cells also include, for example, cardiac muscle cells or cardiomyocytes, and cells of the cardiac vasculatures, such as cells of a coronary artery or vein. Other non-limiting examples of cardiac cells include epithelial cells, endothelial cells, fibroblasts, cardiac stem or progenitor cells, cardiac conducting cells and cardiac pacemaking cells that constitute the cardiac muscle, blood vessels and cardiac cell supporting structure. Cardiac cells may be derived from stem cells, including, for example, embryonic stem cells or induced pluripotent stem cells.

[0247] In some embodiments, the cell is a cardiomyocyte.

[0248] In some embodiments, the cell is an induced pluripotent stem cell (iPSC). In some embodiments, a cell is an iPSC-derived cardiomyocyte.

[0249] In some embodiments, provided are methods of contacting the cell with any vector (e.g., an rAAV virus or virion) described herein. In some embodiments, the cell is a cardiac cell. In some embodiments, the cell is a cardiomyocyte. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0250] In some embodiments, provided are methods of contacting a tissue with any vector (e.g., an rAAV virus or virion) described herein. In some embodiments, the tissue is cardiac tissue. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0251] In some embodiments, provided are methods of contacting an organ with any vector (e.g., an rAAV virus or virion) described herein. In some embodiments, the organ is heart. In some embodiments, the heart is diseased or at risk of disease. In some embodiments, the heart has borderline or reduced ejection fraction. In some embodiments, the heart has a normal ejection fraction. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0252] In some embodiments, provided are cell therapy compositions comprising any cell described herein. Therapeutic Methods

[0253] In some aspects, provided are methods for preventing and / or treating a disease or condition in a subject in need thereof. Subjects who are suitable for theAttorney Docket No. TENA-054 / 01WO 334682-2486 compositions and / or methods of the present technology include individuals, e.g., mammalian subjects, such as humans, non-human primates, domestic mammals, experimental non-human mammalian subjects such as mice, rats, etc. In some embodiments, the subject is a human. In some embodiments, the method comprises overexpressing SYNPO2LA in a cardiac cell (e.g., a cardiomyocyte) in the subject, for example, by administering to the subject a therapeutically effective amount of (i) a vector (e.g., an AAV vector) comprising a polynucleotide encoding a SYNPO2LA polypeptide operatively linked to a cardiac-specific promoter, (ii) a recombinant virion (e.g., a rAAV virus or virion) comprising the vector, or (iii) or a cell comprising the vector, as described herein.

[0254] In some embodiments, the disease or condition is a heart disease. In some embodiments, the heart disease is arrhythmia or atrial fibrillation, which is characterized by an irregular and often rapid heart rhythm. In some embodiment, the atrial fibrillation is familial atrial fibrillation. In some embodiments, the atrial fibrillation is non-familial atrial fibrillation. In some embodiments, the atrial fibrillation is one developed after a previous heart disease, for example, an ischemic heart disease.

[0255] In some embodiments, the subject has or is at risk of having atrial fibrillation, for example, has one or more risk factors contributing to the development of atrial fibrillation. Non-limiting risk factors for atrial fibrillation include age (e.g., older than 50 years), male gender, European ancestry, alcohol use, smoking, high levels of stress, high blood pressure, obesity, diabetes, sleep apnea, chronic obstructive pulmonary disease (COPD), thyroid disease (e.g., hyperthyroidism), chronic kidney disease, and heart- related risk factors such as ischemic heart disease, heart failure, coronary artery disease, cardiomyopathy, pericarditis, congenital heart disease, previous heart surgery, and left atrial or ventricular enlargement. In some embodiments, the subject has had a prior heart disease (e.g., ischemic heart disease, heart failure, coronary artery disease, cardiomyopathy, pericarditis, and / or congenital heart disease).

[0256] In some embodiments, the subject has a genetic variant that increases the risk for atrial fibrillation, for example, has a genetic variant in the SYNPO2L gene that leads to increased expression of SYNPO2LB and / or decreased expression of SYNPO2LA. In some embodiments, the subject has a single-nucleotide polymorphismAttorney Docket No. TENA-054 / 01WO 334682-2486 (SNP) in the SYNPO2L gene that leads to increased expression of SYNPO2LB and / or decreased expression of SYNPO2LA. In some embodiments, the SNP is rs766868752.

[0257] In some embodiments, the subject has atrial fibrillation; prolonged QT; prolonged action potential duration (APD); increased frequency of premature atrial contractions; greater variation in beat period; decreased calcium transient amplitude, calcium influx rates, and / or calcium reuptake; decreased twitch force and / or contraction velocity; greater variation in twitch force and timing; decreased relaxation velocity and / or times; decreased LATS2 protein expression; decreased YAP phosphorylation; and / or decreased transcription of one or more targets of YAP. Non-limiting examples of targets of YAP include TAZ, PTK2, LATS1, YAP1, MITF, USP9X, CDH8, ITGB5, ITGA5, ITGA9, AMOT, KDR, TGFB2, ITGA2, MYOD1, ITGA1, CDH19, KLF4, CDH11, CDH11.1, CCN2, CCN1, ITGAX, and CTNNA1.

[0258] In some embodiments, the subject has one or more symptoms associated with atrial fibrillation selected from irregular heartbeat or arrhythmia, heart palpitation, angina or chest pain, dizziness or lightheadedness, swelling, fainting, fatigue or weakness, and / or shortness of breath. In some embodiments, the heart disease is cardiomyopathy, including, for example, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), idiopathic DCM, arrhythmogenic cardiomyopathy (ACM), and arrhythmogenic right ventricular cardiomyopathy (ARVC). In some embodiments, the heart disease is heart failure, including, for example, heart failure with reduced ejection fraction and ischemic heart failure. In some embodiments, the heart disease is arrhythmia, including, for example, atrial and / or ventricular arrhythmia and malignant ventricular arrhythmia. In some embodiments, the heart disease is cardiomyopathy associated with a pulmonary embolus, venous thrombosis, myocardial infarction, transient ischemic attack, peripheral vascular disorder, atherosclerosis, ischemic cardiac disease, other myocardial injury or vascular disease, and / or cardiac diseases associated with myocardial tissue hypercontractility, such as heart failure related to left ventricular hypercontractility.

[0259] In some embodiments, the pharmaceutical composition for use in the method can be administered to the subject by systemic application (such as parenteral application), for example, by intravenous (e.g., by IV infusion), intra-arterial, or intraperitoneal delivery. In some embodiments, the pharmaceutical composition (e.g.,Attorney Docket No. TENA-054 / 01WO 334682-2486 rAAV vectors, viruses, or virions) can be delivered by direct administration to the heart tissue.

[0260] In some embodiments, the pharmaceutical composition (e.g., rAAV vectors, viruses, or virions) can be delivered by intracoronary administration. In some embodiments, the administration is by antegrade epicardial coronary artery infusion, e.g., a single infusion over a 10-minute period in a cardiac catheterization laboratory after angiography (percutaneous intracoronary delivery without vessel balloon occlusion) with the use of standard 5F or 6F guide or diagnostic catheters.

[0261] In some embodiments, the pharmaceutical composition (e.g., rAAV vectors, viruses, or virions) can be delivered by direct injection into the heart or cardiac catheterization, or by intracardiac catheter delivery via retrograde coronary sinus infusion (RCSI).

[0262] When direct injection is used, it may be performed either by open-heart surgery or by minimally invasive surgery. In some cases, the pharmaceutical composition (e.g., rAAV vectors, viruses, or virions) can be delivered to the pericardial space by injection or infusion.

[0263] In some embodiments, the amount, concentration, and volume of the pharmaceutical composition that modulates contractile function in myocardial tissue administered to a subject can be controlled and / or optimized to substantially improve the functional parameters of the heart while mitigating adverse side effects.

[0264] The amount of the composition that modulates contractile function administered to myocardial tissue can also be an amount required to result in the detectable expression of a therapeutic protein or nucleic acid in the heart; preserve and / or improve contractile function; delay the emergence of cardiomyopathy or reverse the pathological course of the disease; increase myocyte viability; improve myofilament function; inhibit left ventricular hypertrophy; cardiac hypertrophy regression, normalize systolic and diastolic function in heart; and restore normal cross-bridge behavior at the myofilament level.

[0265] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of about 1×108genome copies per milliliter (GC / mL), about 5×108GC / mL, about 1×109GC / mL, about 5×109GC / mL, about 1×1010GC / mL, aboutAttorney Docket No. TENA-054 / 01WO 334682-2486 5×1010GC / mL, about 1×1011GC / mL, about 5×1011GC / mL, about 1×1012GC / mL, about 5×1012GC / mL, about 5×1013GC / mL, about 1×1014GC / mL, or about 5×1014GC / mL of the rAAV vector, virus, or virion.

[0266] In some embodiments, the method comprises intravenously administering an rAAV vector, virus, or virion at a dose of about 3×1012GC / mL, about 3×1013GC / mL, about 1×1014GC / mL, or about 3×1014GC / mL of the rAAV vector, virus, or virion.

[0267] In some embodiments, the method comprises administering, by localized delivery to the heart, an rAAV vector, virus, or virion at a dose of about 3×1011GC / mL, about 3×1012GC / mL, about 1×1013GC / mL, or about 3×1013GC / mL of the rAAV vector, virus, or virion.

[0268] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of about 1×108viral genomes per milliliter (vg / mL), about 5×108vg / mL, about 1×109vg / mL, about 5×109vg / mL, about 1×1010vg / mL, about 5×1010vg / mL, about 1×1011vg / mL, about 5×1011vg / mL, about 1×1012vg / mL, about 5×1012vg / mL, about 5×1013vg / mL, about 1×1014vg / mL, or about 5×1014vg / mL of the rAAV vector, virus, or virion.

[0269] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of less than about 1×1015viral genomes per milliliter (vg / mL), less than about 5×1014vg / mL, less than about 1×1014vg / mL, less than about 5×1013vg / mL, less than about 1×1013vg / mL, less than about 5×1012vg / mL, less than about 1×1012vg / mL, less than about 5×1011vg / mL, or less than about 1×1011vg / mL of the rAAV vector, virus, or virion.

[0270] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of less than about 1×1014viral genomes per milliliter (vg / mL) or less than about 1×1013vg / mL of the rAAV vector, virus, or virion.

[0271] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of from about 1×1011viral genomes per milliliter (vg / mL) to about 1×1015vg / mL, from about 1×1011vg / mL to about 1×1014vg / mL, from about 1×1012vg / mL to about 1×1014vg / mL, from about 1×1012vg / mL to about 1×1013vg / mL, or from about 1×1012vg / mL to about 6×1013vg / mL of the rAAV vector, virus, or virion.Attorney Docket No. TENA-054 / 01WO 334682-2486

[0272] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at any dose or dose range of the disclosure between the values referenced herein.

[0273] In some embodiments, the method comprises intravenously administering an rAAV vector, virus, or virion at a dose of about 1×1012vg / mL, about 3×1012vg / mL, about 6×1012vg / mL, or about 9×1012vg / mL of the rAAV vector, virus, or virion.

[0274] In some embodiments, the method comprises administering, by localized delivery to the heart, an rAAV vector, virus, or virion at a dose of about 1×1012vg / mL, about 3×1012vg / mL, about 6×1012vg / mL, or about 9×1012vg / mL of the rAAV vector, virus, or virion.

[0275] Genome copies per milliliter can be determined by quantitative polymerase change reaction (qPCR) using a standard curve generated with a reference sample having a known concentration of the polynucleotide genome of the virus. For AAV, the reference sample used is often the transfer plasmid used in generation of the rAAV virion, but other reference samples may be used.

[0276] Alternatively, the concentration of a viral vector can be determined by measuring the titer of the vector on a cell line. Viral titer is typically expressed as viral particles (vp) per unit volume (e.g., vp / mL). In various embodiments, the pharmaceutical composition comprises about 1×108viral particles per milliliter (vp / mL), about 5×108vp / mL, about 1×109vp / mL, about 5×109vp / mL, about 1×1010vp / mL, about 5×1010vp / mL, about 1×1011vp / mL, about 5×1011vp / mL, about 1×1012vp / mL, about 5×1012vp / mL, about 5×1013vp / mL, about 1×1014vp / mL, or about 5×1014of the rAAV vector, virus, or virion.

[0277] The vector, virus, or virion administered to the subject can be traced by a variety of methods. For example, recombinant viruses labeled with or expressing a marker (such as green fluorescent protein, or beta-galactosidase) can readily be detected. The recombinant viruses may be engineered to cause the target cell to express a marker protein, such as a surface-expressed protein or a fluorescent protein. Alternatively, the infection of target cells with recombinant viruses can be detected by their expression of a cell marker that is not expressed by the animal employed for testing (for example, a human-specific antigen when injecting cells into an experimental animal). The presence and phenotype of the target cells can be assessed by fluorescenceAttorney Docket No. TENA-054 / 01WO 334682-2486 microscopy (e.g., for green fluorescent protein, or beta-galactosidase), by immunohistochemistry (e.g., using an antibody against a human antigen), by ELISA (using an antibody against a human antigen), or by RT-PCR analysis using primers and hybridization conditions that cause amplification to be specific for RNA indicative of a cardiac phenotype.

[0278] In some embodiments, the vector, virus, or virion, or a pharmaceutical composition containing the same, is administered to the subject once a day, twice a day, three times a day, or four times a day for a period of about 1 day, about 2 days, about 3 days, about 5 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, or more than about 5 years. In some embodiments, the vector, virus, or virion, or a pharmaceutical composition containing the same, is administered every day, every other day, 3 times a week, every third day, weekly, biweekly (i.e., every other week), every third week, monthly, every other month, every third month, every fourth month, every fifth month, every sixth month, every ninth month, every year, every 18 months, every 2 years, every 5 years, every 10 years, or every 20 years. In some embodiments, the dose regimens listed above could be repeated after a period of about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, or more than about 5 years. In some embodiments, the schedule of administration is a hybrid of these periods, for example, the vector, virus, or virion, or a pharmaceutical composition containing the same, is administered a number of times a week and that pattern is repeated a number of times a month every month or every second, third, fourth, fifth, or sixth month, and treatment according to that pattern is continued for part of a year to several years, as set out above. In some embodiments, the vector, virus, or virion, or a pharmaceutical composition containing the same, is administered in a cycle of a number or administrations over a week or two weeks, and the cycle is repeated at spaced intervals over a number of months or years, as set out above. In some embodiments treatment is continued until disease is eliminated, until no further improvement is achieved, or as long as the disease does not progress. In someAttorney Docket No. TENA-054 / 01WO 334682-2486 embodiments, a disease or condition within a subject to be treated can be monitored to evaluate the effectiveness of the treatment using any appropriate method known to a skilled artisan.

[0279] In some embodiments, the vector, virus, or virion, or a pharmaceutical composition containing the same, is administered over a predetermined time period. Alternatively, the vector, virus, or virion, or a pharmaceutical composition containing the same, is administered until a particular therapeutic benchmark is reached. In some embodiments, the methods provided herein further include a step of evaluating one or more therapeutic benchmarks in the subject to determine whether to continue administration of the treatment.

[0280] In some embodiments, the method results in increased expression of SYNPO2LA in a cardiac cell (e.g., a cardiomyocyte) in the subject, for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%, compared to baseline or a control subject who has not received the therapy.

[0281] In some embodiments, the method results in decreased expression of SYNPO2LB in a cardiac cell (e.g., a cardiomyocyte) in the subject, for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, compared to baseline or a control subject who has not received the therapy.

[0282] In some embodiments, the method restores or improves cardiac function in the subject, and / or restores or improves contractile function of the heart in the subject. In some embodiments, the method restores or improves atrial contractility in the subject, for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, compared to baseline or a control subject who has not received the therapy. In some embodiments, the method restores or improves cardiac conduction in the subject, for example, by at least 10%, at least 15%,Attorney Docket No. TENA-054 / 01WO 334682-2486 at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200%, compared to baseline or a control subject who has not received the therapy. In some embodiments, the atrial contractility and / or cardiac conduction is measured by action potential duration (APD), triangulation ratio, and / or beat period. In some embodiments, the atrial contractility and / or cardiac conduction is measured by APD90corrected for beat rate according to Fridericia’s method, beat period variation, twitch force, contraction velocity, twitch force CoV, twitch interval irregularity, relaxation velocity, relaxation / contraction velocity, or RT90.

[0283] In some embodiments, the method increases LATS2 protein level; increases YAP phosphorylation; and / or increases transcription of one or more targets of YAP. Non- limiting examples of targets of YAP include TAZ, PTK2, LATS1, YAP1, MITF, USP9X, CDH8, ITGB5, ITGA5, ITGA9, AMOT, KDR, TGFB2, ITGA2, MYOD1, ITGA1, CDH19, KLF4, CDH11, CDH11.1, CCN2, CCN1, ITGAX, and CTNNA1.

[0284] In some embodiments, the method improves and / or reduces one or more symptoms associated with atrial fibrillation, including, for example, irregular heartbeat or arrhythmia, heart palpitation, angina or chest pain, dizziness or lightheadedness, swelling, fainting, fatigue or weakness, and shortness of breath.

[0285] In some embodiments, the method prevents and / or treats one or more diseases or conditions for which risks are increased by atrial fibrillation, including, for example, blood clot, stroke, heart failure, and dementia.

[0286] In some embodiments, the method increases life span or prevents an increase in mortality over time in the subject with atrial fibrillation. EXAMPLES Example 1. Finnish splice variant confers significant risk for atrial fibrillation

[0287] This study shows that a genetic variant resulting in reduced levels of synaptopodin 2 like (SYNPO2L) gene, isoform A (SYNPO2LA) is associated with increased risks of atrial fibrillation. Rs766868752 (reference A; alternate C; minor allele frequency (MAF) 0.0013) is a rare Finnish splice variant that is strongly associated withAttorney Docket No. TENA-054 / 01WO 334682-2486 increased risk of atrial fibrillation (RR 3.5, 2.20-5.48; p=9.9e-08) first reported in 2021 (FIG.1). Example 2. iPSC cardiomyocytes with Finnish splice variant have altered electrophysiological function

[0288] A human induced pluripotent stem cell (iPSC) line homozygous for the Finnish splice variant, and an isogenic wild-type control iPSC line, were generated and differentiated into high-purity ventricular and atrial iPSC cardiomyocytes using established protocols and cell lines were generated according to established protocols (Lian et al. Proc. Natl. Acad. Sci. U.S.A.2012; 109(27):E1848-E1857; and Cyganek et al. JCI Insight.2018; 3(12):e99941). Cell lines comprising the rs766868752 variant are hereafter referred to as “SYNPO2L Mut+ / +”, and isogenic wild-type control cell lines are hereafter referred to as “WTC”.

[0289] In SYNPO2L Mut+ / +ventricular iPSC cardiomyocytes, mRNA and protein levels of SYNPO2LA were greatly reduced, while the protein level of isoform B (SYNPO2LB) was increased when measured by qPCR and quantitative Western blotting (FIG.2A), compared with WTC. Similarly, SYNPO2L Mut+ / +atrial iPSC cardiomyocytes had significantly reduced levels of SYNPO2LA mRNA and protein (FIG.2B) compared with WTC.

[0290] The electrophysiological properties of SYNPO2L Mut+ / +atrial iPSC cardiomyocytes and WTC were assessed using multi-electrode arrays (MEA). By day 14 of culture, SYNPO2L Mut+ / +atrial iPSC cardiomyocytes exhibited distinct differences in their action potentials compared to WTC atrial iPSC cardiomyocytes. For example, SYNPO2L Mut+ / +atrial iPSC cardiomyocytes exhibited an extended plateau phase and a longer time to repolarization (FIG. 3A). Quantitative analysis confirmed significantly prolonged APDs (FIG. 3B), lower field potential spike amplitudes (FIG. 3C), and decreased spike slopes in SYNPO2L Mut+ / +atrial iPSC cardiomyocytes, in comparison to WTC atrial iPSC cardiomyocytes (FIG. 3D). In comparison, SYNPO2L Mut+ / +ventricular iPSC cardiomyocytes displayed only mild differences in the same electrophysiological parameters (FIGs.3E-3G).

[0291] Increased APDs are indicative of QT prolongation, which has been closely linked to an increased risk of arrhythmias and atrial fibrillation. To confirm thisAttorney Docket No. TENA-054 / 01WO 334682-2486 observation, an extended pacing of the cells was conducted at 2.1 Hz for a total of 90 minutes with 3-minute-long recording periods every 15 minutes. SYNPO2L Mut+ / +atrial iPSC cardiomyocytes exhibited greater beat period variation compared with WTC atrial iPSC cardiomyocytes (FIG. 3H). Furthermore, SYNPO2L Mut+ / +atrial hiPSC cardiomyocytes displayed aberrant beats in 23.8% of timepoints measured, compared to no aberrant beats produced by WTC atrial iPSC cardiomyocytes (p=0.007). Table 4 presents the number of wells producing extra beats, out of the total number of wells, at each time point. Notably, an increased frequency of premature atrial contractions is a recognized risk factor for atrial fibrillation. Table 4. Aberrant beats in atrial hiPSC cardiomyocytes

[0292] Intracellular calcium dynamics were measured in SYNPO2L Mut+ / +atrial iPSC cardiomyocytes and WTC atrial iPSC cardiomyocytes during spontaneous beating, using live imaging of Fluo-4 AM, a fluorescent Ca2+-sensitive dye. SYNPO2L Mut+ / +atrial iPSC cardiomyocytes exhibited an overall decrease in calcium transient amplitude (FIG. 4A and 4B), decreased rates of calcium influx (FIG.4C), and decreased rates of calcium reuptake (FIG. 4D), compared with WTC atrial iPSC cardiomyocytes. In comparison, SYNPO2L Mut+ / +ventricular iPSC cardiomyocytes displayed only mild differences in the same calcium handling parameters, compared with WTC ventricular iPSC cardiomyocytes (FIGS.4E-G).

[0293] RNA sequencing was performed to characterize the transcriptional response of SYNPO2L Mut+ / +atrial iPSC cardiomyocytes compared with WTC atrial iPSC cardiomyocytes (FIG.4H). Downregulation was observed of KCNJ2 and KCNA4, both of which code for potassium ion channels that are key regulators of membrane potentialAttorney Docket No. TENA-054 / 01WO 334682-2486 repolarization and are known to increase arrhythmic risk when their function is disrupted. Additionally, several critical Ca2+handling genes were significantly upregulated, including CACNA1C, ATP2A2, and RYR2. Similar to the aforementioned potassium channels, these calcium channels and receptor play important roles in maintaining proper excitation-contraction coupling and aberrations in their expression or function have been demonstrated to lead to arrhythmogenic events. GJA1 was found to be downregulated in atrial iPSC cardiomyocytes. Dysregulation or remodeling of Cx43 has been observed in patients with atrial fibrillation and in animal models of atrial fibrillation, and overexpression of Cx43 in large animal studies has been found to maintain atrial conduction and prevent persistent atrial fibrillation. These electrophysiological and transcriptional results suggest that SYNPO2L Mut+ / +atrial iPSC cardiomyocytes, which have lower levels of SYNPO2L_A relative to isogenic controls, display a variety of conduction abnormalities strongly consistent with a pro-arrhythmic phenotype. Example 3. Impaired contractility of SYNPO2L Mut+ / +atrial engineered heart tissues.

[0294] SYNPO2L Mut+ / +and WTC atrial iPSC cardiomyocytes were each formed into engineered heart tissues (EHTs) and cultured for 21 days prior to assessment of contractile properties. SYNPO2L Mut+ / +atrial iPSC cardiomyocytes exhibited dramatically lower twitch force and contraction velocities compared with WTC, during both spontaneous beating (FIG.5A) and when paced at 1.5Hz (FIG. 5B). SYNPO2L Mut+ / + EHTs also exhibited greater beat-to-beat variations in twitch force and timing compared to WTC EHTs, during spontaneous beating (FIG. 5A) and when paced at 1.5Hz (FIG.5B). Notably, greater beat-to-beat variations in twitch force and timing are both indicative of arrhythmic behavior.

[0295] Important differences were also observed in the diastolic properties of SYNPO2L Mut+ / + EHTs, which exhibited significantly decreased relaxation velocities and values for RT90 during both spontaneous beating (FIG.5C) when paced at 1.5Hz (FIG.5D). In contrast, EHTs comprised of ventricular hiPSC-CMs carrying the splice mutant displayed more subtle variations in contractile function compared to WTC under both un-paced and paced conditions (FIGs.5E-5H). Transcriptomic analysis of the atrial EHTs showed dramatic downregulation of TTN and ACTN2 in the mutant tissues (FIGS. 5I and 5J). In combination with the electrophysiological readouts, the EHT data overallAttorney Docket No. TENA-054 / 01WO 334682-2486 suggest that functional deficits may arise from the haploinsufficiency of a specific SYNPO2L isoform and that the severity of these effects may be heart chamber specific. Example 4. LATS stabilization and YAP signaling in SYNPO2L Mut+ / +atrial iPSC cardiomyocytes.

[0296] The paralog of SYNPO2L has been observed to interact with the LATS1 / 2 complex to modulate YAP / TAZ signaling in other model systems, and the role of YAP / TAZ in the HIPPO signaling pathway in cardiac development and disease is well recognized. Without wishing to be bound by theory, SYNPO2L_A may interact with the LATS1 / 2 complex to modulate YAP / TAZ signaling in hiPSC-CMs. Interestingly, while assessment of LATS1 and LATS2 transcript expression in atrial cardiomyocytes suggested no appreciable difference between WTC and SYNPO2L Mut+ / + cells (FIGS. 6A and 6B), immunoblotting showed that at the protein level, LATS2 expression was significantly decreased in SYNPO2L Mut+ / + cells (FIG.6C). Without wishing to be bound by theory, these data are consistent with a role for SYNPO2L_A in stabilizing LATS2.

[0297] Immunoblotting also suggested that while YAP levels were indistinguishable between the splice mutant and WTC cardiomyocytes (FIG.6D), phosphorylated YAP (pYAP) was significantly lower in the splice mutant (FIG. 6E). Without wishing to be bound by theory, this decrease in phosphorylation may suggest the degradation of LATS2. Additionally, in transcriptional analyses of WTC and SYNPO2L Mut+ / + EHTs, we noted broad and significant changes in known downstream targets of YAP transcriptional modulation (FIG.7, Table 5). Without wishing to be bound by theory, these data may support a SYNPO2L isoform specific effect upon binding LATS2 to modulate downstream targets of YAP / TAZ transcriptional regulation. Table 5. Transcriptional response of downstream targets of YAP signaling in SYNPO2L Mut+ / +atrial iPSC-CMs.Attorney Docket No. TENA-054 / 01WO 334682-2486Example 5. SYNPO2L_A overexpression reduces pro-arrhythmic dysfunction

[0298] To examine the effects of restoring intracellular expression of SYNPO2L_A, vectors were constructed with either an expression cassette containing the SYNPO2L_A isoform under the control of the human TNNT2 promoter (AAV:SYNPO2L_A), or no open reading frame (AAV: No ORF). These cassettes are shown in FIG.8A. Each expression cassette was packaged each into a AAV9-derivative capsid.

[0299] After 30 days of culture, SYNPO2L Mut+ / +atrial iPSC cardiomyocyte monolayers were treated with wither AAV:SYNPO2L_A or AAV: No ORF at a 5K multiplicity of infection (MOI) for 24 hours. By 7 days post-transduction, expression of SYNPO2L_A in SYNPO2L Mut+ / +atrial iPSC cardiomyocytes treated with AAV:SYNPO2L_A (SYNPO2L Mut+ / ++ AAV:SYNPO2L_A) had reached approximately 9-fold that of WTC (FIG. 8B). Electrophysiological measurements taken by multi- electrode arrays (MEA) 20 days post-transduction showed significant restoration of action potential durations (FIGS.9A and 9B), to levels observed in WTC cells treated with AAV:No ORF. Only partial restoration of spike amplitudes (FIG.9C) and spike slopes (FIG.9D) was observed.

[0300] Furthermore, SYNPO2L Mut+ / +atrial iPSC cardiomyocytes treated with AAV:SYNPO2L_A exhibited a 21.4% reduction in aberrant beats over a 90-minute pacing period (p=0.006), compared to SYNPO2L Mut+ / +atrial iPSC cardiomyocytes treated withAttorney Docket No. TENA-054 / 01WO 334682-2486 AAV: No ORF. Table 6 presents the number of wells producing extra beats, out of the total number of wells, at each time point. Table 6. Aberrant beats in SYNPO2L Mut+ / +atrial hiPSC cardiomyocytes

[0301] In a parallel study with EHTs, treatment of SYNPO2L Mut+ / +atrial EHTs with the same dose of AAV:SYNPO2L_A partially improved pre-treatment abnormalities in twitch force (FIG.10A) and contractile velocity (FIG.10B). Notably, metrics for arrhythmic contraction such as twitch force CoVs (FIG.10C) and twitch interval irregularity (FIG. 10D), were fully normalized in SYNPO2L Mut+ / +atrial EHTs treated with AAV:SYNPO2L_A, compared to WTC EHTs treated with AAV:No ORF. Diastolic function, in particular relaxation velocities (FIGS.10E and 10F) and relaxation times (FIG. 10G), also exhibited improvements in SYNPO2L Mut+ / +atrial EHTs treated with AAV:SYNPO2L_A, compared to WTC EHTs treated with AAV:No ORF; however, the degree of rescue was not statistically significant at 20 days post-transfection. Example 6. Common variant associated with decreased isoform B and increased isoform A is related to protection from atrial fibrillation and related phenotypes

[0302] This study shows that a common variant, rs3740293, results in decreased expression of SYNPO2LB and decreases risks for atrial fibrillation and related phenotypes. The C allele of rs3740293 is observed to be a splice quantitative trait locus (sQTL) whereby in RNA sequencing measurements of gene expression across 54 tissues in 1000 individuals, the C allele was strongly associated with decreased expression of the B isoform and increased expression of the A isoform (FIG.3). The same C allele was also strongly associated with protection from atrial fibrillation and heartAttorney Docket No. TENA-054 / 01WO 334682-2486 failure, decreased PR interval and pulse rate, and increased left ventricular mass in genome-wide association studies (see Table 7 below). Table 7. Lower SYNPO2LB levels conferred by rs3740293 improves risk for atrial fibrillation and related phenotypesAdapted from public database at https: / / cvd.hugeamp.org accessed 12 / 10 / 23

[0303] Another common variant, rs60632610, which is closely linked (R2=0.94) to rs3740293, shows that decreased isoform B is protective against the development of atrial fibrillation after ischemic heart disease (FIG. 12). In individuals from the UK Biobank database, three groups of individuals were defined with a different AF origin, where a diagnosis of AF occurred more than 1 month after a primary diagnosis of: a) non-ischemic heart failure, b) mitral or aortic valve disease, or c) ischemic heart disease. Individuals were matched for age, BMI, and sex at diagnosis. Individuals with the T allele of rs60632610 (corresponding to the C allele of rs3740293, lower expression of isoform B, and higher expression of SYNPO2L_A in the left atrium) were protected against development of atrial fibrillation specifically amongst individuals who experienced myocardial infarction (MI) (HR of 0.92 per allele, p=5.27e-6). In contrast, individuals with the T allele of Rs60632610 who were experiencing atrial fibrillation after a diagnosis of valvular disease, or in non-ischemic heart failure, were not protected.

[0304] Genetic co-localization analysis identified rs60632610 as a most likely causal SNP in the SYNPO2L region co-localizing with dilated cardiomyopathy, atrial fibrillation, heart failure, left ventricular ejection fraction, systolic and diastolic blood pressure and short axis pulmonary artery diameter (Table 8).Attorney Docket No. TENA-054 / 01WO 334682-2486 Table 8. A splice QTL for SYNPO2L SNP rs60632610 co-localizes with a variety of related traits.

[0305] Taken together, these data suggest that a therapy conferring increased levels of SYNPO2LA (e.g., an AAV-based therapeutics encoding the SYNPO2LA gene) might be a useful treatment for prevention or treatment of atrial fibrillation after a diagnosis of ischemic heart disease, and that the treatment effect could be maximized by selecting a patient population using genetics to identify individuals who carry the common genetic variants that lower SYNPO2LA levels and / or increase SYNPO2LB levels. Additionally, the AAV-based SYNPO2LA therapeutics may be useful for treating yet to be identified protein coding variations in SYNPO2L which may increase the risk of atrial fibrillation, and / or for preventing or treating other clinical forms of atrial fibrillation that are not a consequence of ischemic heart disease.

[0306] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

Attorney Docket No. TENA-054 / 01WO 334682-2486 CLAIMS I / We claim:

1. A method of preventing and / or treating atrial fibrillation in a subject in need thereof, the method comprising overexpressing synaptopodin 2 like (SYNPO2L), isoform A (SYNPO2LA) in a cardiac cell in the subject.

2. The method of claim 1, wherein the overexpressing is by administering to the subject a therapeutically effective amount of (i) a vector comprising a polynucleotide encoding a SYNPO2LA polypeptide operatively linked to a cardiac-specific promoter, (ii) a recombinant virion comprising the vector, or (iii) or a cell comprising the vector.

3. The method of claim 1 or 2, wherein the subject has had a prior heart disease selected from ischemic heart disease, heart failure, coronary artery disease, cardiomyopathy, pericarditis, and congenital heart disease.

4. The method of claim 1 or 2, wherein the subject has a genetic variant in the SYNPO2L gene that leads to increased expression of SYNPO2L, isoform B (SYNPO2LB) and / or decreased expression of SYNPO2LA.

5. The method of claim 4, wherein the genetic variant in the in the SYNPO2L gene comprises a single-nucleotide polymorphism (SNP).

6. The method of claim 5, wherein the SNP is rs766868752.

7. The method of any one of claims 2-6, wherein the SYNPO2LA polypeptide comprises an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2, and / or the polynucleotide encoding the SYNPO2LA polypeptide comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1.Attorney Docket No. TENA-054 / 01WO 334682-2486 8. The method of any one of claims 2-7, wherein the cardiac-specific promoter is a troponin T (TNNT2) promoter or a chimeric variant thereof.

9. The method of claim 8, wherein the TNNT2 promoter or chimeric variant thereof comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 5-16.

10. The method of any one of claims 2-9, wherein the vector is an adeno-associated virus (AAV) vector; and / or the recombinant virion is a recombinant AAV (rAAV) virion.

11. The method of claim 10, wherein the AAV vector is an AAV9 vector; and / or the rAAV virion comprises an AAV9 capsid protein or a variant thereof described herein.

12. The method of any one of claim 2-11, wherein the administering results in: a) increased expression of SYNPO2LA in the cardiac cell in the subject, optionally wherein the cardiac cell is a cardiomyocyte; b) decreased expression of SYNPO2LB in the cardiac cell in the subject, optionally wherein the cardiac cell is a cardiomyocyte; c) increased atrial contractility; d) increased cardiac conduction; e) improvement in one or more symptoms associated with atrial fibrillation selected from irregular heartbeat or arrhythmia, heart palpitation, angina or chest pain, dizziness or lightheadedness, swelling, fainting, fatigue or weakness, and shortness of breath; and / or f) reduced risk for blood clot, stroke, heart failure, and / or dementia.

13. The method of any one of claims 2-12, wherein the administering is systemic administration or local administration to the heart.

14. The method of claim 13, wherein the systemic administration is intravenous administration.Attorney Docket No. TENA-054 / 01WO 334682-2486 15. The method of claim 13, wherein the local administration is by direct injection into the heart or cardiac tissue, intracoronary administration, or retrograde coronary sinus infusion.

16. A method of increasing SYNPO2L_A expression in cardiac cells of a subject in need thereof comprising administration of a therapeutically effective amount of: (i) a vector comprising a polynucleotide encoding a SYNPO2LA polypeptide operatively linked to a cardiac-specific promoter, (ii) a recombinant virion comprising the vector, or (iii) a cell comprising the vector.

17. The method of claim 16, wherein the subject has the rs766868752 SNP in the SYNPO2L gene.

18. The method of claim 16 or claim 17, wherein the SYNPO2LA polypeptide comprises an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2, and / or the polynucleotide encoding the SYNPO2LA polypeptide comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

1.

19. The method of any one of claims 16-18, wherein the cardiac-specific promoter is a troponin T (TNNT2) promoter or a chimeric variant thereof.

20. The method of claim 19, wherein the TNNT2 promoter or chimeric variant thereof comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 5-16.

21. The method of any one of claims 16-20, wherein the vector is an adeno-associated virus (AAV) vector; and / or the recombinant virion is a recombinant AAV (rAAV) virion.Attorney Docket No. TENA-054 / 01WO 334682-2486 22. The method of claim 21, wherein the AAV vector is an AAV9 vector; and / or the rAAV virion comprises an AAV9 capsid protein or a variant thereof described herein.

23. A method of selecting a subject for treatment with (i) a vector comprising a polynucleotide encoding a SYNPO2LA polypeptide operatively linked to a cardiac- specific promoter; (ii) a recombinant virion comprising the vector; or (iii) a cell comprising the vector, wherein the subject has the rs766868752 SNP in the SYNPO2L gene.

24. The method of claim 23, comprising: i) determining the presence of the rs766868752 SNP in the SYNPO2L gene in the subject; and ii) administering to the subject a therapeutically effective amount of the vector, virion, or cell if the SNP is present in the SYNPO2L gene of the subject.

25. The method of claim 23 or 24, wherein the subject suffers from atrial fibrillation; prolonged QT; prolonged action potential duration (APD); increased frequency of premature atrial contractions; greater variation in beat period; decreased calcium transient amplitude, calcium influx rates, and / or calcium reuptake; decreased twitch force and / or contraction velocity; greater variation in twitch force and timing; decreased relaxation velocity and / or times; decreased LATS2 protein expression; and / or decreased YAP phosphorylation.

26. The method of any one of claims 23-25, wherein the subject has one or more symptoms associated with atrial fibrillation selected from irregular heartbeat or arrhythmia, heart palpitation, angina or chest pain, dizziness or lightheadedness, swelling, fainting, fatigue or weakness, and / or shortness of breath.

27. The method of any one of claims 23-26, wherein the SYNPO2LA polypeptide comprises an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2, and / or the polynucleotide encoding the SYNPO2LA polypeptide comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%,Attorney Docket No. TENA-054 / 01WO 334682-2486 at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

1.

28. The method of any one of claims 23-27, wherein the cardiac-specific promoter is a troponin T (TNNT2) promoter or a chimeric variant thereof.

29. The method of claim 28, wherein the TNNT2 promoter or chimeric variant thereof comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 5-16.

30. The method of any one of claims 23-29, wherein the vector is an adeno-associated virus (AAV) vector; and / or the recombinant virion is a recombinant AAV (rAAV) virion.

31. The method of claim 30, wherein the AAV vector is an AAV9 vector; and / or the rAAV virion comprises an AAV9 capsid protein or a variant thereof described herein.

32. An expression cassette comprising a polynucleotide encoding a SYNPO2L_A polypeptide operably linked to a TNNT2 promoter or chimeric variant thereof.

33. The expression cassette of claim 32, wherein the SYNPO2L_A polypeptide is a wild type human SYNPO2L_A polypeptide comprising the amino acid sequence of SEQ ID NO:

2.

34. The expression cassette of claim 33, wherein the polynucleotide encoding the wild type human SYNPO2L_A polypeptide is a codon-optimized polynucleotide.

35. The expression cassette of claim 34, wherein the polynucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO:

1.

36. The expression cassette of any one of claims 32-35, wherein the TNNT2 promoter or chimeric variant thereof is a human TNNT2 promoter.Attorney Docket No. TENA-054 / 01WO 334682-2486 37. The expression cassette of any one of claims 32-36, wherein the TNNT2 promoter or chimeric variant thereof comprises a sequence sharing at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to any one of SEQ ID NOs: 5-16.

38. The expression cassette of claim 36 or claim 37, wherein the human TNNT2 promoter is a wild-type human TNNT2 promoter of 600 bp, optionally having 100% identity to SEQ ID NO:

5.

39. The expression cassette of any one of claims 32-38, wherein the human TNNT2 promoter has a sequence sharing at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to SEQ ID NO:

7.

40. The expression cassette of claim 39, wherein the human TNNT2 promoter is a modified human TNNT2 promoter of 350 bp to 450 bp, or 350 bp to 500 bp.

41. The expression cassette of claim 39, wherein the human TNNT2 promoter is a modified human TNNT2 promoter of about 400 bp of SEQ ID NO:

7.

42. The expression cassette of any one of claims 32-41, wherein the expression cassette comprises a polyadenylation sequence.

43. The expression cassette of claim 42, wherein the polyadenylation sequence is a bGH poly(A) sequence.

44. The expression cassette of claim 43, wherein the bGH poly(a) has a sequence at least 50%, at least 75%, at least 80%, at least 95% or 100% identical to SEQ ID NO:

23.

45. A vector comprising the expression cassette of any one of claims 32-44, optionally wherein the vector is an adeno-associated virus (AAV) vector, a retroviral vector, or a lentiviral vector.

46. The vector of claim 45, wherein the vector is an AAV vector, optionally wherein the AAV vector is an AAV9 vector.

47. The vector of claim 46, wherein the vector comprises any capsid protein described herein, optionally wherein the capsid protein is a wild-type AAV9 capsid protein or an engineered variant thereof.Attorney Docket No. TENA-054 / 01WO 334682-2486 48. A pharmaceutical composition comprising the vector of any one of claims 45-47, further comprising a pharmaceutically acceptable carrier, diluent, or excipient.

49. A method of treating a disease caused by or associated with a genetic variant in the SYNPO2L gene that leads to increased expression of SYNPO2L, isoform B (SYNPO2LB) and / or decreased expression of SYNPO2LA, in a subject in need thereof, the method comprising administering to the subject the vector of any one of claims 45- 47 or the pharmaceutical composition of claim 48.

50. The method of claim 49, wherein the genetic variant in the in the SYNPO2L gene comprises a single-nucleotide polymorphism (SNP).

51. The method of claim 50, wherein the SNP is rs766868752.

52. The method of any one of claims 49-51, wherein the subject has had a prior heart disease selected from ischemic heart disease, heart failure, coronary artery disease, cardiomyopathy, pericarditis, and congenital heart disease.

53. The method of any one of claims 49-52, wherein the administering results in: (a) increased expression of SYNPO2LA in the cardiac cell in the subject, optionally wherein the cardiac cell is a cardiomyocyte; (b) decreased expression of SYNPO2LB in the cardiac cell in the subject, optionally wherein the cardiac cell is a cardiomyocyte; (c) increased atrial contractility; (d) increased cardiac conduction; (e) improvement in one or more symptoms associated with atrial fibrillation selected from irregular heartbeat or arrhythmia, heart palpitation, angina or chest pain, dizziness or lightheadedness, swelling, fainting, fatigue or weakness, and shortness of breath; and / or (f) reduced risk for blood clot, stroke, heart failure, and / or dementia.

54. The method of any one of claims 49-53, wherein the administering is systemic administration or local administration to the heart.Attorney Docket No. TENA-054 / 01WO 334682-2486 55. The method of claim 54, wherein the systemic administration is intravenous administration.

56. The method of claim 54, wherein the local administration is by direct injection into the heart or cardiac tissue, intracoronary administration, or retrograde coronary sinus infusion.

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