WO2025235491A1

WO2025235491A1 - Recombinant aav for treatment of cardiac diseases

Info

Publication number: WO2025235491A1
Application number: PCT/US2025/027963
Authority: WO
Inventors: John Scott REECE-HOYES; Lisa M. STANEK; Roberto CALCEDO DEL HOYO; Thomas M. EDWARDS; Giridhar MURLIDHARAN
Original assignee: Affinia Therapeutics Inc
Current assignee: Affinia Therapeutics Inc
Priority date: 2024-05-07
Filing date: 2025-05-06
Publication date: 2025-11-13
Anticipated expiration: 2026-11-07

Abstract

The disclosure pertains to recombinant adeno-associated viruses (rAAVs) comprising a capsid with a targeting peptide for delivering a polynucleotide encoding a BAGS protein or MYBPC3 protein to the heart. Further provided are pharmaceutical compositions comprising the rAAVs and methods for treating cardiac diseases using the rAAVs and pharmaceutical compositions.

Description

RECOMBINANT AAV FOR TREATMENT OF CARDIAC DISEASES

1 . CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. provisional application nos. 63/643,759, filed May 7, 2024, and 63/723,079, filed November 20, 2024, the contents of each of which are incorporated herein in their entireties by reference thereto.

2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on April 28, 2025, is named 63134WO_CRF_sequencelisting.xml and is 365,386 bytes in size.

3. BACKGROUND

[0003] Mutations in B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) and myosin-binding protein C, cardiac-type (MYBPC3) protein are associated with cardiac diseases. For example, BAG3 mutations are associated with dilated cardiomyopathy and MYBPC3 mutations are associated with hypertrophic cardiomyopathy. There is an unmet need for gene therapies for treating cardiac diseases, including those associated with BAG3 and MYBPC3 mutations.

4. SUMMARY

[0004] The present disclosure addresses the need in the art for gene therapies for treating cardiac diseases, including dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), and provides recombinant adeno-associated viruses (rAAVs) having improved cardiac tropism as compared to rAAV with wild-type AAV9 capsid proteins.

[0005] In some aspects, the disclosure provides a rAAV comprising (a) a capsid with a modified capsid protein having a targeting peptide within variable region VIII (VR VIII) comprising the amino acid sequence X1X2X3RGDX7X8X9X10, where X1X2X3 is SAQ, ASS, ENK, or ENR, and X7, Xs, X9, and X10 are independently selected from any amino acid residue, and (b) a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein or a myosin-binding protein C, cardiac-type (MYBPC3) protein. An exemplary targeting peptide where X1X2X3 is SAQ is SEQ ID NO:1 . An exemplary targeting peptide where X1X2X3 is ASS is SEQ ID NO:2. An exemplary targeting peptide where X1X2X3 is ENK is SEQ ID NO:3. An exemplary targeting peptide where X1X2X3 is ENR is SEQ ID NO:4.

[0006] In some aspects, the disclosure provides a rAAV comprising (a) a capsid with a modified capsid protein having a targeting peptide within variable region VIII (VR VIII) comprising the amino acid sequence X1X2X3RGDRGQI (SEQ ID NO:12), X1X2X3RGDFNNL (SEQ ID NO:13), or X1X2X3RGDYTSM (SEQ ID NO:14), where X1X2X3 are independently selected from any amino acid residue, and (b) a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein or a myosin-binding protein C, cardiactype (MYBPC3) protein.

[0007] In some aspects, the disclosure provides a rAAV comprising (a) a capsid with a modified capsid protein having a targeting peptide within variable region VIII (VR VIII), the targeting peptide comprising the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 and (b) a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein or a myosin-binding protein C, cardiactype (MYBPC3) protein.

[0008] Exemplary amino acid sequences of modified capsid proteins having a targeting peptide are set forth in SEQ ID NOS:8-11 . SEQ ID NO:8 has a targeting peptide comprising the amino acid sequence of SEQ ID NO:1 (SEQ ID NO:8 also has a peptide segment comprising the amino acid sequence of SEQ ID NO:5); SEQ ID NO:9 has a targeting peptide comprising the amino acid sequence of SEQ ID NO:2 (SEQ ID NO:9 also has a peptide segment comprising the amino acid sequence of SEQ ID NO:6); SEQ ID NQ:10 has a targeting peptide comprising the amino acid sequence of SEQ ID NO:3 (SEQ ID NQ:10 also has a peptide segment comprising the amino acid sequence of SEQ ID NO:7); and SEQ ID NO:11 has a targeting peptide comprising the amino acid sequence of SEQ ID NO:4 (SEQ ID NO:11 also has a peptide segment comprising the amino acid sequence of SEQ ID NO:7). A VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:8 is sometimes referred to herein as “Variant 1”; a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:9 is sometimes referred to herein as “Variant 2”; a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NQ:10 is sometimes referred to herein as “Variant 3”; and a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:11 is sometimes referred to herein as “Variant 4.”

[0009] In one aspect, the disclosure provides a rAAV comprising (a) a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:8, and VP2 and VP3 portions thereof and (b) a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a BAG3 protein comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NQ:100.

[0010] In another aspect, the disclosure provides a rAAV comprising (a) a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:9, and VP2 and VP3 portions thereof and (b) a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a BAG3 protein comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NQ:100.

[0011] In another aspect, the disclosure provides a rAAV comprising (a) a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NQ:10, and VP2 and VP3 portions thereof and (b) a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a BAG3 protein comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NQ:100.

[0012] In another aspect, the disclosure provides a rAAV comprising (a) a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:11 , and VP2 and VP3 portions thereof and (b) a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a BAG3 protein comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID N0:100.

[0013] In another aspect, the disclosure provides a rAAV comprising a capsid and (b) a polynucleotide encapsulated by the capsid and comprising a promoter sequence as described herein operably linked a coding sequence encoding a BAG3 protein comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NQ:100.

[0014] Exemplary BAG3 protein coding sequences that can be used in the rAAV of the disclosure are set forth in SEQ ID NQS:101-103.

[0015] In another aspect, the disclosure provides a rAAV comprising (a) a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:8, and VP2 and VP3 portions thereof and (b) a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a MYBPC3 protein comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NQ:200 or SEQ ID NQ:201 .

[0016] In another aspect, the disclosure provides a rAAV comprising (a) a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:9, and VP2 and VP3 portions thereof and (b) a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a MYBPC3 protein comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NQ:200 or SEQ ID NQ:201 .

[0017] In another aspect, the disclosure provides a rAAV comprising (a) a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NQ:10, and VP2 and VP3 portions thereof and (b) a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a MYBPC3 protein comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NQ:200 or SEQ ID NQ:201 .

[0018] In another aspect, the disclosure provides a rAAV comprising (a) a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:11 , and VP2 and VP3 portions thereof and (b) a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a MYBPC3 protein comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID N0:200 or SEQ ID NO:201 .

[0019] In another aspect, the disclosure provides a rAAV comprising a capsid and (b) a polynucleotide encapsulated by the capsid and comprising a promoter sequence as described herein operably linked a coding sequence encoding a MYBPC3 protein comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID N0:200 or SEQ ID NQ:201 .

[0020] Exemplary MYBPC3 protein coding sequences that can be used in the rAAV of the disclosure are set forth in SEQ ID NQS:202-203.

[0021] In another aspect, the disclosure provides polynucleotides that can be included in an rAAV. Exemplary polynucleotides are provided in SEQ ID NQS:800-810 and SEQ ID NQS:900-910.

[0022] Further exemplary features of rAAV and polynucleotides of the disclosure are described in Section 6.2 (e.g., features of exemplary capsid polypeptides are described in Section 6.2.1 and features of exemplary polynucleotides as described in Section 6.2.2) and specific embodiments 1 to 440, infra.

[0023] The disclosure further provides host cells capable of producing the rAAV of the disclosure, for example as described in Section 6.3 and specific embodiments 443 to 445, /nfra.

[0024] The disclosure further provides pharmaceutical compositions comprising the rAAV of the disclosure and a pharmaceutically acceptable excipient and unit doses thereof, for example as described in Section 6.4 and specific embodiments 441 to 442, infra.

[0025] The disclosure further provides methods of transferring polynucleotides encoding BAG3 or MYBPC3 to the heart of a subject using the rAAV, pharmaceutical compositions, and unit doses of the disclosure, and provides methods of treating subjects having or at risk of a cardiac disease, for example a subject having DCM or HCM. The disclosure further provides the rAAVs, pharmaceutical compositions, and unit doses of the disclosure for use in methods of the disclosure. Exemplary methods of the disclosure, and rAAVs, pharmaceutical compositions, and unit doses of the disclosure for use in such methods are described in Section 6.5 and specific embodiments 446 to 515, infra.

5. BRIEF DESCRIPTION OF THE FIGURES

[0026] FIGS. 1A and 1B show the eGFP expression in heart tissues of mice that received rAAV having Variant 1 capsid proteins or rAAV having wild-type AAV9 capsid proteins expressing eGFP at multiple dose levels, with FIG. 1 A showing the immunohistochemistry (IHC) staining in heart tissue sections of mice and FIG. 1B showing the RT-ddPCR results of heart tissues of mice (Example 1). [0027] FIG. 2 shows eGFP mRNA expression in the heart tissues of mice that received rAAV having Variant 1 capsid proteins or rAAV having Variant 4 capsid proteins at post-injection day 28, day 60, and day 90 (Example 1).

[0028] FIGS. 3A, 3B, and 3C show the transcription efficiency of eGFP in heart tissues of mice that received rAAV having Variant 1 capsid proteins, rAAV having Variant 4 capsid proteins, or rAAV with wild-type AAV9 capsid proteins at multiple dose levels, with FIG. 3A showing the GFP mRNA level in heart tissues of mice, FIG. 3B showing the GFP DNA level in heart tissues of mice, and FIG. 3C showing the mRNA/DNA ratio of Variant 1 and Variant 4 over AAV9 (Example 1).

[0029] FIG. 4 shows the immunohistochemistry (IHC) staining in heart, liver, and DRG tissue sections of NHPs that received rAAVs with Variant 1 , Variant 4, or wild-type AAV9 capsid proteins expressing eGFP at high dose level (Example 2).

[0030] FIG. 5 shows the immunohistochemistry (IHC) staining in heart tissue sections of NHPs received rAAVs with Variant 1 , Variant 4, or wild-type AAV9 capsid proteins expressing eGFP at low dose level and high dose level (Example 2).

[0031] FIGS. 6A and 6B show the percentage of GFP+ cells in different heart regions of NHPs received rAAVs with Variant 1 , Variant 4, or wild-type AAV9 capsid proteins expressing eGFP, with FIG. 6A showing the percentage of GFP+ cells with low dose of rAAVs with Variant 1 , Variant 4, or wild-type AAV9 capsid proteins and FIG. 6B showing the percentage of GFP+ cells with high dose of rAAVs with Variant 1 , Variant 4, or wild-type AAV9 capsid proteins (Example 2).

[0032] FIGS. 7A and 7B show the GFP mRNA level in different heart regions of NHPs received rAAVs with Variant 1 , Variant 4, or wild-type AAV9 capsid proteins expressing eGFP, with FIG. 7A showing the GFP mRNA level with low dose of rAAVs with Variant 1 , Variant 4, or wild-type AAV9 capsid proteins and FIG. 7B showing the GFP mRNA level with high dose of rAAVs with Variant 1 , Variant 4, or wild-type AAV9 capsid proteins (Example 2).

[0033] FIG. 8 shows the immunohistochemistry (IHC) staining in different heart region sections of NHPs that received rAAVs with Variant 1 or wild-type AAV9 capsid proteins expressing eGFP at low dose level (Example 2).

[0034] FIG. 9 schematically illustrates the rAAV genome of Example 3.

[0035] FIG. 10 schematically illustrates the timeline of the study of Example 3.

[0036] FIG. 11 shows HA tagged BAG3 expression in the heart as measured by IHC (Example 3).

[0037] FIG. 12 shows HA tagged BAG3 expression in the heart as measured by JESS™ automated western blot (Example 3). [0038] FIG. 13 shows HA tagged IHC staining of heart (left panel), liver (middle panel) and quadriceps muscle (right panel) (Example 3). All images shown at 4X magnification.

[0039] FIGS. 14A-14D show ejection fraction of mice of Example 3 at various time points (FIGS. 14A and 14C) and nine weeks post-surgery (three weeks post-AAV injection) (FIGS. 14B and 14D). Values are mean ± SEM. Ml untreated ejection fraction data (from a previous characterization study) at 3, 6, and 9 weeks post Ml surgery are included for reference. *p < 0.05 compared with Mi-Untreated (ANOVA).

[0040] FIGS. 15A-15D show ejection fraction (FIG. 15A), fractional shortening (FIG. 15B), treatment condition-related changes to left ventricular posterior wall (LVPW) thickness (FIG. 15C) at 2 WPI, and change in ejection fraction percentage from 0 WPI to 2WPI (FIG. 15D) in hearts of WT and BAG3 cKO mice that received rAAVs with Variant 3 capsid proteins encapsulating polynucleotides comprising a BAG3 transgene under the control of CK8e promoter and comprising or lacking OPRE (Example 4).

[0041] FIGS. 16A-16R show vector genome levels in hearts (FIG. 16A) and livers (FIG. 16B) at 8 days WPI, vector genome levels in hearts (FIG. 16C) and livers (FIG. 16D) at 28 days WPI, BAG3 RNA levels in hearts (FIG. 16E) and livers (FIG. 16F) at 8 days WPI, BAG3 RNA levels in hearts (FIG. 16G) and livers (FIG. 16H) at 28 days WPI of WT mice that received rAAVs with Variant 3 capsid proteins encapsulating a polynucleotide comprising a BAG3 transgene under the control of a MLC2 promoter; and ejection fraction between 0 WPI and 4 WPI (FIG. 161) and ejection fraction (FIG. 16J), fractional shortening (FIG. 16K), left ventricular interior diameter (FIG. 16L) and left ventricular posterior wall thickness (FIG. 16M) at 4 WPI, as well as BAG3 RNA expression (FIG. 16N), vector genome (FIG. 160), BAG3 protein levels (FIG. 16P) in hearts and HA tagged IHC staining (FIG. 16Q) and protein quantification (FIG. 16R) in hearts of BAG3 cKO mice that received rAAVs with Variant 3 or AAV9 capsid proteins encapsulating polynucleotides comprising a BAG3 transgene under the control of MLC2 promoter (Example 5).

[0042] FIGS. 17A-17D show vector genome levels in hearts (FIG. 17A) and livers (FIG. 17B), BAG3 RNA levels in hearts (FIG. 17C) and livers (FIG. 17D) of WT mice that received rAAVs with Variant 3 capsid proteins encapsulating polynucleotides comprising a BAG3 transgene under the control of MLC2 promoter or Ck8e promoter (Example 6).

[0043] FIGS. 18A-18G show BAG3 RNA levels (FIG. 18A), vector genome levels (FIG. 18B), BAG3 protein levels (FIGS. 18C and 18D), HA tagged IHC staining (FIG. 18E and 18F) and protein quantification (FIG. 18G) in hearts of WT mice that received rAAVs with Variant 1 capsid proteins encapsulating polynucleotides comprising a BAG3 transgene under the control of MLC2, TNNT2-400, TNNT2-600, aMHC, or BAG3 promoter (Example 7).

[0044] FIGS. 19A-19D show ejection fraction (FIG. 19A), vector genome (FIG. 19B), BAG3 RNA (FIG. 19C) and BAG3 protein levels (FIG. 19D) in hearts of mice that received rAAVs with Variant 1 capsid proteins encapsulating polynucleotides comprising a BAG3 transgene under the control of a BAG3 promoter in a mouse model of myocardial infarction (Ml) (Example 8). [0045] FIGS. 20A-20C show BAG3 RNA expression (FIG. 20A), BAG3 protein levels (FIG. 20B), and vector genome levels (FIG. 20C) in hearts of WT mice 14, 28, and 60 days after they received 2e13 vg/kg or 1e14 vg/kg rAAV comprising Variant 1 capsid proteins encapsulating a polynucleotide comprising a BAG3 transgene under the control of a BAG3 promoter (Example 9).

[0046] FIGS. 21A-21D show BAG3 RNA expression (FIG. 21 A) and vector genome levels (FIG. 21 B) in hearts of WT mice that received 2e12 vg/kg or 6.32e11 vg/kg rAAV comprising Variant 4 capsid proteins encapsulating a polynucleotide comprising a BAG3 transgene under the control of a MLC2 promoter; and BAG3 RNA expression (FIG. 21 C) and vector genome levels (FIG. 21 D) in hearts of WT mice that received 2e12 vg/kg rAAV comprising Variant 1 or Variant 4 capsid proteins encapsulating polynucleotides comprising a BAG3 transgene under the control of a MLC2 promoter or a BAG3 promoter (Example 10).

[0047] FIGS. 22A-22B show BAG3 RNA expression (FIG. 21 A) and vector genome levels (FIG. 21 B) in hearts of WT mice that received a 2e13 vg/kg or 6.32e12 vg/kg dose of rAAV comprising Variant 2 capsid proteins encapsulating a polynucleotide comprising a BAG3 transgene under the control of a MLC2 promoter (Example 11).

[0048] FIGS. 23A-23K show ejection fraction (FIG. 23A), fractional shortening (FIG. 23B), corrected left ventricle (LV) mass per body weight (FIG. 23C), LV mass (FIG. 23D), left ventricle wall diameter (FIG. 23E), vector genome levels (FIG. 23F), BAG3 RNA expression (FIG. 23G), BAG3 protein levels (FIG. 23H-23K) in BAG3 cKO mice that received a 2e13 vg/kg dose of rAAV comprising Variant 2 or Variant 4 capsid proteins encapsulating a polynucleotide comprising a BAG3 transgene under the control of a MLC2 promoter (Example 12).

[0049] FIGS. 24A-24H show MYBPC3 RNA expression in hearts (FIG. 24A) and quadriceps (FIG. 24B), vector genome levels in hearts (FIG. 24C) and quadriceps (FIG. 24D), RNA levels normalized for vector genome copy numbers (FIG. 24E and FIG. 24F) and MYPBC3 protein expression in hearts (FIG. 24G and FIG. 24H) of WT mice that received a 2e12 vg/kg or 2e13 vg/kg dose of rAAV comprising Variant 1 capsid proteins encapsulating polynucleotides comprising a MYBPC3 transgene under the control of a TNNT2, MHCK7, Ck8e, or MSEC-725A promoter (Example 13).

[0050] FIGS. 25A-25E show ejection fraction overtime (FIG. 25A) and 22 weeks post-dosing (FIG. 25B), vector genome (FIG. 25C) and MYBPC3 RNA expression (FIG. 25D) and MYBPC3 protein (FIG. 25E) levels in hearts of MYBPC3 KI mice that received a 6.32e12 vg/kg or 2e13 vg/kg dose of rAAV comprising Variant 1 capsid proteins encapsulating a polynucleotide comprising a MYBPC3 transgene under the control of a Ck8e promoter or a 2e13 vg/kg dose of rAAV comprising AAV9 capsid proteins encapsulating a polynucleotide comprising a MYBPC3 transgene under the control of a TNNT promoter (Example 14).

[0051] FIGS. 26A-26C show vector genome (FIG. 26A), MYBPC3 RNA expression (FIG. 26B), and

MYPBC3 protein levels in WT mice that received a 2e12 vg/kg or 2e13 vg/kg dose of rAAV comprising Variant 1 capsid proteins encapsulating a polynucleotide comprising a MYBPC3 transgene under the control of a TNNT2, MHCK7, Ck8e, or MSEC-725A promoter or rAAV comprising Variant 4 capsid proteins encapsulating a polynucleotides comprising a MYBPC3 transgene under the control of a MYH7, CSRP3, HRC, and MYOZ2 promoter (Example 15).

[0052] FIGS. 27A-27S show ejection fraction overtime (FIG. 27A), fractional shortening overtime (FIG. 27B), left ventricle internal diameter during systole over time (FIG. 27C), left ventricle internal diameter during diastole over time (FIG. 27D), ejection fraction at 14 WPI (FIG. 27E), and ejection fraction in hearts of male and female mice at 11 WPI (FIGS. 27F-I), vector genome levels at 14 WPI (FIGS. 27J and 27K), and MYBPC3 RNA expression at 14 WPI (FIGS. 27L and 27M), MYPC3 protein levels at 14 WPI (FIGS. 27N-27Q), and HA tagged IHC staining (FIGS. 27R and 27S) in hearts of MYBPC3 KI mice that received a 2e13 vg/kg or 1e14 vg/kg dose of rAAV comprising Variant 2 capsid proteins encapsulating a polynucleotide comprising a MYBPC3 transgene under the control of Ck8e promoter (Example 16).

[0053] FIGS. 28A-28G show ejection fraction at 6 WPI (FIG. 28A) and overtime (FIG. 28B), fractional shortening over time (FIG. 28C), left ventricle internal diameter during systole overtime (FIG. 28D), left ventricle internal diameter during diastole overtime (FIG. 28E), LV mass overtime (FIG. 28F) and at 6WPI (FIG. 28G) of MYBPC3 KI mice that received a 2e13 vg/kg or 1e14 vg/kg dose of rAAV comprising Variant 2 capsid proteins encapsulating a polynucleotide comprising a mouse MYBPC3 transgene under the control of Ck8e promoter (Example 17).

[0054] FIGS. 29A-29E show MYBPC3 RNA expression (FIG. 29A) and vector genome levels (FIG. 29B) at 28 and 70 days WPI, MYBPC3 protein expression (FIGS. 29C-29E) in MYBPC3 KI mice that received a 2e13 vg/kg or 6e13 vg/kg dose of rAAV comprising Variant 2 capsid proteins encapsulating a polynucleotide comprising a mouse MYBPC3 transgene under the control of a Ck8e promoter (Example 18).

[0055] FIGS. 30A-30G show ejection fraction over time (FIG. 30A), at 2WPI (FIG. 30B), and at 6 WPI (FIG. 30C), vector genome levels (FIG. 30D), mouse MYBPC3 RNA expression (FIG. 30E), human MYBPC3 RNA expression (FIG. 30F), and MYBPC3-HA Tag protein levels (FIG. 30G) in KI mice that received a 6e12 vg/kg, 3e13 vg/kg or 1e14 vg/kg dose of rAAV comprising Variant 3 capsid proteins encapsulating a polynucleotide comprising a mouse or a human MYBPC3 transgene under the control of a Ck8e promoter (Example 19).

[0056] FIGS. 31A-31I show vector genome levels (FIG. 31 A) and human MYBPC3 RNA expression (FIG. 31 B) in young and adult mice, LV mass overtime (FIG. 31 C), vector genome levels (FIG. 31 D), human MYBPC3 RNA expression (FIG. 31 E), and human MYBPC3 protein levels (FIG. 31 F), mouse MYBPC3 RNA expression (FIG. 31 G), MYBPC3-Tag protein (FIG. 31 H) and MYBPC3 total protein (FIG. 311) levels in hearts of HOM or HET MYBPC3 KI mice that received a 6e12 vg/kg, 3e13 vg/kg or 1e14 vg/kg dose of rAAV comprising Variant 3 capsid proteins encapsulating a polynucleotide comprising a mouse or a human MYBPC3 transgene under the control of a Ck8e promoter (Example 20). [0057] FIGS. 32A-32C show ejection fraction over time (FIG. 32A) and at 11 WPI (FIG 32B) and at 11 and 13 WPI (FIG. 32C) in HET MYBPC3 KI mice that received a 6.32e12 vg/kg, 3e13 vg/kg or 1e14 vg/kg dose of rAAV comprising Variant 3 capsid proteins encapsulating a polynucleotide comprising a mouse or a human MYBPC3 transgene under the control of a Ck8e promoter (Example 21).

[0058] FIGS. 33A-33B show vector genome levels (FIG. 33A) and MYPBC3 RNA expression (FIG. 33B) in hearts of WT or MYBPC3 KI mice that received rAAVs comprising Variant 1 , Variant 2, or Variant 3 capsid proteins and encapsulating polynucleotides comprising an eGFP coding sequence under the control of CK8e promoter (Example 22).

[0059] FIG. 34A-34E show BAG3 protein concentration obtained with JESS™ automated western blot (FIG. 34A) and ELISA (FIG. 34B), GFP levels obtained by flow cytometry (FIG. 34C) in C2C12 cells and BAG3 protein expression (FIGS. 34D and 34E) in iPSC-CM cells that were transduced with rAAV comprising Variant 3 capsid proteins comprising BAG3 under the control of MLC-2, CK8e, or CBh promoter (Example 23).

[0060] FIGS. 35A-35B show vector genome copies (VGC) per mL in whole blood (Example 24). FIG. 35A: data for groups administered rAAV at 1 e14 vg/kg. FIG. 35B: data for groups administered rAAV at 3e13vg/kg. Pre-administration measurements at day -28 and day 1 were below the limit of detection.

[0061] FIGS. 36A-36B show vector genome copies (VGC) per mL in plasma (Example 24). FIG. 36A: data for groups administered rAAV at 1e14 vg/kg. FIG. 36B: data for groups administered rAAV at 3e13vg/kg. Pre-administration measurements at day -28 and day 1 were below the limit of detection.

[0062] FIGS. 37A-37B show vector genome copies (VGC) per ng in blood cell pellets (Example 24). FIG. 37A: data for groups administered rAAV at 1 e14 vg/kg. FIG. 37B: data for groups administered rAAV at 3e13vg/kg. Pre-administration measurements at day -28 and day 1 were below the limit of detection.

[0063] FIGS. 38A-38B show vector shedding in urine (FIG. 38A) and feces (FIG. 38B) following administration of rAAV having different capsid proteins to NHPs (Example 24). Data in FIGS. 38A-38B are for groups administered rAAV at 1e14 vg/kg. Pre-administration measurement at day -28 was below limit of detection.

[0064] FIGS. 39A-39D show levels of liver enzymes ALT (FIG. 39A and FIG. 39B) and AST (FIG. 39C and FIG. 39D) in NHPs administered rAAV having different capsid proteins (Example 24) (study day 8). Dashed line indicates maximum levels reported in healthy cynomolgus macaques in Park et al., 2016, Lab Anim Res 32(2):79-86. Data in FIG. 39A and 39C are for groups administered rAAV at 1e14 vg/kg. Data in FIG. 39B and 39D are for groups administered rAAV at 3e13 vg/kg.

[0065] FIGS. 40A-40B show vector genome number per diploid genome number (FIG. 40A) and mRNA expression (FIG. 40B) in cervical DRG from NHPs administered rAAV having different capsid proteins (Example 24). Squares at 3e13 vg/kg (21 -day in life) added from another study to increase n value of AAV9 treatments when samples available. Black datapoints indicate NHP sacrificed early.

[0066] FIGS. 41A-41 B show vector genome number per diploid genome number (FIG. 41 A) and mRNA expression (FIG. 41 B) in lumbar DRG from NHPs administered rAAV having different capsid proteins (Example 24). Squares at 3e13 vg/kg (21 -day in life) added from another study to increase n value of AAV9 treatments when samples available.

[0067] FIGS. 42A-42B show vector genome number per diploid genome number (FIG. 42A) and mRNA expression (FIG. 42B) in liver from NHPs administered rAAV having different capsid proteins (Example 24). Squares at 3e13 vg/kg (21 -day in life) added from another study to increase n value of AAV9 treatments when samples available. Black datapoints indicate NHP sacrificed early.

[0068] FIGS. 43A-43B show percent GFP positive cells in liver (FIG. 43A) and DRG (FIG. 43B) from NHPs administered rAAV having different capsid proteins (Example 24). Black datapoint in FIG. 43A indicates NHP sacrificed early. In FIG. 43B, a dot is mean for an animal; a bar is median.

[0069] FIGS. 44A-44B show seroprevalence of antibodies reactive with capsid proteins in two sets of human sera donor samples (Example 25). FIG. 44A shows seroprevalence of antibodies reactive with AAV9 capsid protein, Variant 1 capsid protein, and Variant 4 capsid protein in a first set of donor samples (n=50). FIG. 44B shows seroprevalence of antibodies reactive with AAV9 capsid protein, Variant 2 capsid protein, and Variant 3 capsid protein in a second set of donor samples (n=100).

[0070] FIG. 45 shows H&E and HA-tagged IHC staining in heart of a cynomolgus macaque that received a 3e13 vg/kg dose of rAAV with Variant 3 capsid proteins encapsulating polynucleotides comprising a BAG3 transgene under the control of a CK8e promoter (Example 26).

[0071] FIG. 46 shows H&E and HA-tagged IHC staining in heart of two cynomolgus macaques that received a 1e13 vg/kg dose of rAAV with Variant 3 capsid proteins encapsulating polynucleotides comprising a BAG3 transgene under the control of a CK8e promoter (Example 26).

[0072] FIGS. 47A-47D show ejection fraction (FIG. 47A), fractional shortening (FIG. 47B), and treatment condition-related changes to left ventricle internal diameter during systole and diastole (LVIDs & LVIDd) (FIG. 47C and FIG. 47D, respectively) in hearts of WT and BAG3 cKO mice that received rAAVs with Variant 3 capsid proteins encapsulating polynucleotides comprising a BAG3 transgene under the control of CK8e promoter and comprising or lacking OPRE (Example 27). Data obtained 11 weeks post-injection, except for group administered 6.32e11 dose, for which data was obtained 12 weeks post-injection.

[0073] FIGS. 48A-48F show vector genome levels in the left ventricle (FIG. 48A) and in the right ventricle, IVS and quadriceps tissues (FIG. 48B), RNA levels in left ventricle (FIG. 48C) and in the right ventricle, IVS and quadriceps tissues (FIG. 48D), and changes in BAG3 protein levels normalized by the expression in (FIG. 48E) or in comparison to (FIG. 48F) naive heart tissues of cynomolgus macaque that received rAAVs with Variant 3 capsid proteins encapsulating polynucleotides comprising a BAG3 transgene under the control of a CK8e promoter (Example 28).

6. DETAILED DESCRIPTION

6.1. Definitions

[0074] As used herein, the following terms are intended to have the following meanings:

[0075] A, An, The: As used herein, the term “a”, “an”, “the” and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.

[0076] AAV: AAV is adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise.

[0077] AAV capsid protein: The term “AAV capsid protein” or simply “capsid protein” refers to a VP1 , VP2, or VP3 capsid protein. A capsid protein that is modified as compared to a naturally occurring or synthetic / artificial capsid protein capsid protein is referred to as a “modified AAV capsid protein” or simply “modified capsid protein” or “variant capsid protein.” The naturally occurring or synthetic / artificial capsid protein against which a modified AAV capsid protein is referred to herein as a “reference” capsid protein. In some embodiments, the AAV capsid protein is a modified capsid protein of AAV9; AAV2; AAV1 ; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV hu.68; AAV10; AAV5; AAV3- 3; AAV4-4; AAV1-A; hu.46-A; hu.48-A; hu.44-A; hu.43-A; AAV6-A; hu.34-B; hu.47-B; hu.29-B; rh.63-B; hu.56-B; hu.45-B; rh.57-B; rh.35-B; rh.58-B; rh.28-B; rh.51-B; rh.19-B; rh.49-B; rh.52-B; rh.13-B; AAV2-B; rh.20-B; rh.24-B; rh.64-B; hu.27-B; hu.21-B; hu.22-B; hu.23-B; hu.7-C; hu.61-C; rh.56-C; hu. 9-C; hu.54- C; hu.53-C; hu.60-C; hu.55-C; hu.2-C; hu.1-C; hu.18-C; hu.3-C; hu.25-C; hu.15-C; hu.16-C; hu.11-C; hu.10-C; hu.4-C; rh.54-D; rh.48-D; rh.55-D; rh.62-D; AAV7-D; rh.52-E; rh.51-E; hu.39-E; rh.53-E; hu.37- E; rh.43-E; rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; rh74; hu.42-E; rh.57-E; rh.40-E; hu.67-E; hu.17-E; hu.6-E; hu.66-E; rh.38-E; hu.32-F; AAV9/hu; hu.31-F; Anc80; Anc81 ; Anc82; Anc83; Anc84; Anc94;

And 13; Anc126; Anc127; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33;

Anc80L36; Anc80L44; Anc80L1 ; And 10; and Anc80DI. In some embodiments, the AAV capsid protein is a modified capsid protein of AAV9.

[0078] Amino Acid Position: The term “amino acid position” within an AAV capsid protein refers to a position of an amino acid residue in an AAV VP1 protein sequence, counted from the first amino acid at the N terminal. As used herein, the term "amino acid" comprises naturally occurring L- and D- amino acids and artificial, i.e. non-naturally occurring, a-amino acids. Preferably, the amino acid is a naturally occurring amino acid. In preferred embodiments, the amino acid is a naturally occurring L-a-amino acid. For the avoidance of doubt, as used herein, the indication that an insertion site is at amino acid position X means that the targeting peptide is inserted between amino acids X and X+1 , i.e., the targeting peptide is inserted after the amino acid at position X and before the amino acid at position X+1. The position of an amino acid in an AAV capsid protein that “corresponds to” a position in a reference AAV capsid protein can be established by the skilled person by known methods, preferably by aligning the amino acids of the capsid proteins.

[0079] And/or: The term “and/or” means that each one or both or all the components or features of a list are possible variants, especially two or more thereof in an alternative or cumulative way.

[0080] BAG3 Protein: The term “BAG3 protein” refers to human B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) or a functional fragment or functional variant thereof. An exemplary BAG3 protein sequence is provided as UniProt Accession No. 095817. In some embodiments, a BAG3 protein has at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to SEQ ID N0:100 or a functional fragment or functional variant thereof.

[0081] Coding Sequence: The term “coding sequence” is used herein to refer to a specific sequence of nucleotides in a polynucleotide, such as an rAAV genome or mRNA produced thereby, that encodes a polypeptide.

[0082] Effective amount: The term “effective amount” or “therapeutically effective amount” means the amount or quantity of an agent or composition that is sufficient to elicit the required or desired response, or in other words, the amount that is sufficient to elicit an appreciable biological response when administered, e.g., to a subject. Said amount preferably relates to an amount that is therapeutically effective against the progression of a disease or disorder as disclosed herein. It is understood that an “effective amount” or a “therapeutically effective amount” can vary from subject to subject, due to variation in metabolism of an agent, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.

[0083] Expression Regulatory Element: The term “expression regulatory element” or “ERE” as used herein in the context of the rAAV of the disclosure refers to a nucleic acid sequence which is required for expression of a BAG3 or MYBPC3 coding sequence operably linked to the ERE. In some instances, an ERE sequence may be a core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product, for example exon sequences.

[0084] Functional Fragment: The term “functional fragment” in the context of a BAG3 or MYBPC3 protein refers to a biologically functional fragment of full length BAG3 or MYBPC3. As would be understood in the art, a biologically functional fragment is a portion or portions of a full length sequence that retain a biological function of the full length sequence. Biological functions of BAG3 include acting as a co-chaperone for HSP70 and HSC80 chaperone proteins, acting as a nucleotide-exchange factor (NEF) promoting the release of ADP from the HSP70 and HSC70 proteins thereby triggering client/substrate protein release, anti-apoptopic activity, and playing a role in HSF1 nucleocytoplasmic transport. Biological functions of MYBPC3 include modifying the activity of actin-activated myosin ATPase, and modulating cardiac contraction.

[0085] Functional Variant: The term “functional variant” in the context of BAG3 or MYBPC3 refers to various splicing isoforms, variants, fusion proteins, and modified forms of a wild-type BAG3 or MYBPC3 polypeptide or a functional fragment thereof. Such isoforms, bioactive fragments or variants, fusion proteins, and modified forms of the BAG3/MYBPC3 polypeptides retain at least one biological function of the full-length protein.

[0086] Inverted Terminal Repeat: The term “inverted terminal repeat” (or “ITR”) refers to a polynucleotide sequence found at the ends of AAV genomes that form a hairpin, which contributes to the genome’s ability to self-prime (allowing for primase-independent synthesis of the complementary second DNA strand) and provides for encapsidation of the genome into an AAV particle. An ITR can be a wildtype ITR or a variant thereof.

[0087] Liver-Toggle: The terms “liver-toggle mutant”, “liver-toggle mutant of a reference AAV capsid protein” and the like, as used herein, refers to a capsid protein comprising a sequence different from a reference AAV capsid protein by having one or more mutations (e.g., amino acid substitutions) that alter tropism, specificity or distribution in a liver as compared to the reference AAV capsid protein when administered to a mammalian subject (such a sequence difference referred to herein as a “liver toggle mutation”). The mammalian subject can be a human, non-human primate (NHP), mice, rats, birds, rabbits, guinea pigs, hamsters, farm animals (including pigs and sheep), dogs, or cats. Exemplary liver toggle mutations are disclosed in WO2019/217911 and W02021/050614, incorporated by reference in their entireties herein. In some embodiments, the liver toggle mutations comprise (i) an alanine (A) or guanine (G) amino acid residue at an amino acid position corresponding to position 266 in Anc80 VP1 and/or b) a lysine (K) or arginine (R) amino acid residue at an amino acid position corresponding to position 168 in Anc80 VP1 . In other embodiments, a liver-toggle mutant of a reference AAV capsid protein is a capsid protein comprising a sequence different from the reference AAV capsid protein by having an alanine (A) amino acid residue at an amino acid position corresponding to position 267 in AAV9 VP1 protein and a threonine (T) amino acid residue at an amino acid position corresponding to position 269 in AAV9 VP1 . In yet further embodiments, the liver toggle mutations comprise a sequence different from the reference AAV capsid protein by having any combination of (i) an arginine (R) instead of serine (S) at position 446; (ii) an alanine (A) instead of an arginine (R) at position 471 ; and (iii) a threonine (T) or alanine (A) instead of a valine (V) at position 708, in each case numbered according to an AAV2 reference capsid protein (SEQ ID NO:1 of W02021/050614, which is incorporated by reference herein).

[0088] Modification: The term “modification” when in conjunction with an amino acid residue, amino acid residues, or a modified sequence, refers to insertion(s), deletion(s), and/or substitution^).

[0089] MYBPC3 Protein: The term “MYBPC3 protein” refers to human B myosin-binding protein C, cardiac-type (MYBPC3) or a functional fragment or functional variant thereof. An exemplary MYBPC3 protein sequence is provided as UniProt Accession No. Q14896-1 . Another exemplary MYBPC3 protein sequence is provided as UniProt Accession No. Q14896-2. In some embodiments, a MYBPC3 protein has at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to SEQ ID N0:200 or a functional fragment or functional variant thereof. In some embodiments, a MYBPC3 protein has at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to SEQ ID NO:201 or a functional fragment or functional variant thereof.

[0090] Operably Linked: The terms “operably linked” and “operatively linked” refer to the functional relationship of the nucleic acid sequences with regulatory sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences and indicates that two or more DNA segments are joined together such that they function in concert for their intended purposes. For example, operative linkage of nucleic acid sequences, typically DNA, to a regulatory sequence or promoter region refers to the physical and functional relationship between the DNA and the regulatory sequence or promoter such that the transcription of such DNA is initiated from the regulatory sequence or promoter, by an RNA polymerase that specifically recognizes, binds and transcribes the DNA.

[0091] Or: Unless indicated otherwise, an “or” conjunction is intended to be used in its correct sense as a Boolean logical operator, encompassing both the selection of features in the alternative (A or B, where the selection of A is mutually exclusive from B) and the selection of features in conjunction (A or B, where both A and B are selected). In some places in the text, the term “and/or” is used for the same purpose, which shall not be construed to imply that “or” is used with reference to mutually exclusive alternatives.

[0092] Parenteral: The term “parenteral” administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) , or intrasternal injection, or infusion techniques.

[0093] Peptide: The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.

[0094] Peptide Segment: The term “peptide segment” as used herein refers to a part of variable region I (VR I) of an AAV capsid protein comprising 12 amino acids. In preferred embodiments, the peptide segment is positioned between amino acid 250 and 280 of the AAV capsid protein. A modified AAV capsid protein provided herein can include, but does not necessarily include, a peptide segment having a sequence different from the corresponding sequence of a reference AAV capsid protein by having one or more modifications.

[0095] Percent Sequence Identity: The terms “percent sequence identity” (% sequence identity), “percent identical” (% identical) and the like refer to percent sequence identity between two nucleotide sequences or between two amino acid sequences calculated by aligning the two sequences, determining the number of matches of nucleotides or amino acid residues between the two sequences, dividing the number of matches by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues), and multiplying by 100 to arrive at a percent sequence identity value. For calculation of the percent sequence identity (% sequence identity), two or more sequences are aligned using the EMBOSS Needle Pairwise Sequence Alignment software tool based on the Needleman and Wunsch algorithm (available at www.ebi.ac.uk/jdispatcher/psa/emboss_needle) with the following parameters: Matrix: BLOSUM62 (for protein sequences) or DNAfull (for DNA sequences); Gap Open: 10; Gap Extend: 0.5; End Gap Penalty: false; End Gap Open: 10; and End Gap Extend: 0.5.

[0096] Pharmaceutically acceptable carrier: The term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, excipients, stabilizers and adjuvants. For examples of carriers, excipients, stabilizers and adjuvants, see Remington: The Science and Practice of Pharmacy, 22nd Revised Ed., Pharmaceutical Press, 2012.

[0097] rAAV: The abbreviation “rAAV” refers to a recombinant adeno-associated viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide, sometimes referred to herein as a “genome”. rAAV can include a genome that comprises a heterologous polynucleotide (/.e., a polynucleotide other than a wild-type AAV genome), such as a heterologous polynucleotide encoding a gene delivered to a mammalian cell such as the BAG3 or MYBPC3 gene. The heterologous nucleotide is sometimes referred to as a transgene.

[0098] Self-complementary: The term “self-complementary” rAAV vector or genome as used herein means a fully or partially self-complementary rAAV vector or genome, respectively. A “fully self- complementary” rAAV vector refers to a vector containing a genome generated by the absence of a terminal resolution site (TR) from one of the ITRs of the rAAV. The absence of a TR prevents the initiation of replication at the vector terminus where the TR is not present. In general, fully self- complementary rAAV vectors generate single-stranded, inverted repeat genomes, with a wild-type (wt) AAV TR at each end and a mutated TR (mTR) in the middle. Thus, a fully self-complementary rAAV genome is typically a single stranded polynucleotide having, in the 5' to 3' direction, a first ITR sequence, a heterologous sequence (e.g., BAG3 or MYBPC3 coding sequence and/or ERE), a second ITR sequence, a second heterologous sequence that is complementary to the first heterologous sequence, and a third ITR sequence. A “partially self-complementary” rAAV genome refers to a single stranded polynucleotide having, in the 5' to 3' direction or the 3' to 5' direction, a first ITR sequence, a heterologous sequence (e.g., BAG3 or MYBPC3 coding sequence and/or ERE), a second ITR sequence, and a self- complementary region that is complementary to a portion of the heterologous sequence and has a length that is less than the entire length the heterologous sequence.

[0099] Targeting Peptide: The term “targeting peptide” refers to a 10 amino acid sequence within the variable region VIII (VRVIII) of a modified AAV capsid protein introduced by one or more modifications described herein. AAVs comprising a modified capsid protein with a targeting peptide can have localization and distribution in a target cell, tissue or organ (e.g., heart) different from the AAV with a capsid protein without the target peptide.

[0100] Tissue-specific: A “tissue-specific” (e.g., muscle-specific) promoter as used herein refers to a nucleotide sequence which, when operably linked with a polynucleotide encoding a BAG3 protein or a MYBPC3 protein, causes the BAG3 or MYBPC3 protein to be produced in a cell substantially only if the cell is a cell of the tissue type.

[0101] Treatment: The terms “treatment”, “treating”, and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease, condition, or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for a disease or condition and/or adverse effect attributable to the disease or condition. “Treatment” as used herein covers any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition (e.g., arresting its development); or (c) relieving the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms).

[0102] Variable Region: The terms “variable region” or “VR” refer to one or more of nine sequence variable regions (e.g., VRI to VRIX) in an AAV capsid protein previously defined by comparison and alignment of various AAV capsid proteins. See e.g., Govindasamy et al., 2006, J Virol. 80(23):11556-70; Meyer et al., 2019, Elife 22:8:e44707; DiMattia et al., J Virol. 86(12):6947-6958.

[0103] Vector: The terms “vector,” “AAV vector,” and “rAAV vector” refer to an rAAV that comprises a heterologous polynucleotide, e.g., a transgene.

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety

6.2. Recombinant Adeno-Associated Virus

[0105] One aspect of the present disclosure provides a rAAV comprising (a) a capsid with a modified capsid protein as described in PCT publication no. WO 2024/040193 A2 (the contents of which are incorporated herein by reference in their entireties) and (b) a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein or a myosin-binding protein C, cardiac-type (MYBPC3) protein.

[0106] Another aspect of the present disclosure provides an rAAV comprising a capsid protein having a targeting peptide within variable region VIII (VR VIII) comprising the amino acid sequence X1X2X3RGDX7X8X9X10, where X1X2X3 is SAQ, ASS, ENK, or ENR and X₇, X₈, X₉, and X10 are independently selected from any amino acid residue and a polynucleotide encapsulated by the capsid and encoding BAG3 or MYBPC3. [0107] Another aspect of the present disclosure provides a rAAV comprising (a) a capsid with a modified capsid protein having a targeting peptide within variable region VIII (VR VIII) comprising the amino acid sequence X1X2X3RGDRGQI (SEQ ID NO:12), X1X2X3RGDFNNL (SEQ ID NO:13), or X1X2X3RGDYTSM (SEQ ID NO:14), where X1X2X3 are independently selected from any amino acid residue, and a polynucleotide encapsulated by the capsid and encoding BAG3 or MYBPC3.

[0108] Another aspect of the present disclosure provides an rAAV comprising a capsid protein having a targeting peptide within variable region VIII (VR VIII), wherein the targeting peptide comprises the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 and a polynucleotide encapsulated by the capsid and encoding BAG3 or MYBPC3.

6.2.1. Capsid

[0109] In some aspects, the rAAV of the disclosure have a capsid comprising a modified capsid protein having a targeting peptide within VR III. In some embodiments, the targeting peptide comprises an amino acid sequence as set forth in SEQ ID NO:1 . In other embodiments, the targeting peptide comprises an amino acid sequence as set forth in SEQ ID NO:2. In yet other embodiments, the targeting peptide comprises an amino acid sequence as set forth in SEQ ID NO:3. In yet other embodiments, the targeting peptide comprises an amino acid sequence as set forth in SEQ ID NO:4. The targeting peptide can be located between amino acid positions corresponding to positions 585 and 589 of a wild-type AAV9 capsid protein sequence as set forth in SEQ ID NO:32, with the amino acids corresponding to positions 586, 587, and 588 of the wild-type AAV9 capsid protein sequence replaced with the amino acids of the targeting peptide.

[0110] The modified capsid protein can comprise, in addition to a targeting peptide, one or more (e.g., two) liver-toggle mutations as compared to a reference capsid protein (e.g., as compared to the AAV9 capsid protein sequence set forth in SEQ ID NO:32). For example, the modified capsid protein can include an alanine at the amino acid position corresponding to position 267 of SEQ ID NO:32 and/or a threonine at the amino acid position corresponding to position 269 of SEQ ID NO:32. In some embodiments, the modified capsid protein includes an alanine at the amino acid position corresponding to position 267 of SEQ ID NO:32 and a threonine at the amino acid position corresponding to position 269 of SEQ ID NO:32.

[0111] The modified capsid can comprise, in addition to a targeting peptide, a peptide segment within variable region I (VR I), for example at amino acid positions corresponding to positions 262 to 273 of the AAV9 capsid protein sequence set forth in SEQ ID NO:32. The targeting peptide can include one or more liver-toggle mutations. In some embodiments, the peptide segment comprises an amino acid sequence as set forth in SEQ ID NO:5. In other embodiments, the peptide segment comprises an amino acid sequence as set forth in SEQ ID NO:6. In yet other embodiments, the peptide segment comprises an amino acid sequence as set forth in SEQ ID NO:7.

[0112] In some embodiments, the modified peptide comprises a targeting peptide of SEQ ID NO:1 and a peptide segment of SEQ ID NO:5. In other embodiments, the modified peptide comprises a targeting peptide of SEQ ID NO:2 and a peptide segment of SEQ ID NO:6. In yet other embodiments, the modified peptide comprises a targeting peptide of SEQ ID NO:3 and a peptide segment of SEQ ID NO:7. In yet other embodiments, the modified peptide comprises a targeting peptide of SEQ ID NO:4 and a peptide segment of SEQ ID NO:7.

[0113] The rAAV used in various embodiments of the present disclosure comprises a capsid formed with VP1 , VP2 and VP3 capsid proteins. In a particular embodiment, the capsid is formed with VP1 , VP2 and VP3 capsid proteins of a modified capsid protein disclosed herein.

[0114] In some embodiments, VP1 protein has the amino acid sequence of SEQ ID NO:8 (which has a targeting peptide of SEQ ID NO:1 and a peptide segment of SEQ ID NO:5 (which includes liver toggle mutations)). In some embodiments, the VP1 protein comprises a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8. In some embodiments, VP2 and VP3 proteins have a portion of the amino acid sequence of SEQ ID NO:8. In some embodiments, VP2 protein has a sequence corresponding to amino acids 138 to 743 of SEQ ID NO:8 and VP3 protein can have a sequence corresponding to amino acids 203 to 743 of SEQ ID NO:8. In some embodiments, VP2 protein has a sequence corresponding to a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to amino acids 138 to 743 of SEQ ID NO:8 and/or VP3 protein can have a sequence corresponding to a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to amino acids 203 to 743 of SEQ ID NO:8.

[0115] In some embodiments, VP1 protein has the amino acid sequence of SEQ ID NO:9 (which has a targeting peptide of SEQ ID NO:2 and a peptide segment of SEQ ID NO:6). In some embodiments, the VP1 protein comprises a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9. In some embodiments, VP2 and VP3 proteins have a portion of the amino acid sequence of SEQ ID NO:9. In some embodiments, VP2 protein has a sequence corresponding to amino acids 138 to 743 of SEQ ID NO:9 and VP3 protein can have a sequence corresponding to amino acids 203 to 743 of SEQ ID NO:9. In some embodiments, VP2 protein has a sequence corresponding to a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to amino acids 138 to 743 of SEQ ID NO:9 and/or VP3 protein can have a sequence corresponding to a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to amino acids 203 to 743 of SEQ ID NO:9.

[0116] In some embodiments, VP1 protein has the amino acid sequence of SEQ ID NQ:10 (which has a targeting peptide of SEQ ID NO:3 and a peptide segment of SEQ ID NO:7). In some embodiments, the VP1 protein comprises a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NQ:10. In some embodiments, VP2 and VP3 proteins have a portion of the amino acid sequence of SEQ ID NQ:10. In some embodiments, VP2 protein has a sequence corresponding to amino acids 138 to 743 of SEQ ID NQ:10 and VP3 protein can have a sequence corresponding to amino acids 203 to 743 of SEQ ID NQ:10. In some embodiments, VP2 protein has a sequence corresponding to a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to amino acids 138 to 743 of SEQ ID NQ:10 and/or VP3 protein can have a sequence corresponding to a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to amino acids 203 to 743 of

SEQ ID NQ:10.

[0117] In some embodiments, VP1 protein has the amino acid sequence of SEQ ID NO:11 (which has a targeting peptide of SEQ ID NO:4 and a peptide segment of SEQ ID NO:7). In some embodiments, the VP1 protein comprises a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:11 . In some embodiments, VP2 and VP3 proteins have a portion of the amino acid sequence of SEQ ID NO:11. In some embodiments, VP2 protein has a sequence corresponding to amino acids 138 to 743 of SEQ ID NO:11 and VP3 protein can have a sequence corresponding to amino acids 203 to 743 of SEQ ID NO:11 . In some embodiments, VP2 protein has a sequence corresponding to a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to amino acids 138 to 743 of SEQ ID NO:11 and/or VP3 protein can have a sequence corresponding to a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to amino acids 203 to 743 of SEQ ID NO:11.

6.2.2. Polynucleotide

[0118] The rAAV disclosed herein comprises a polynucleotide encapsulated by the capsid. The polynucleotide comprises a sequence encoding a BAG3 protein or a MYBPC3 protein. The coding sequence can be codon optimized, e.g., for expression in human cardiac cells. Alternatively, the coding sequence can be a wild-type sequence.

[0119] The BAG3 protein is preferably a human BAG3 protein, for example having an amino acid sequence of UniProt accession no. 095817, set forth herein as SEQ ID NQ:100. In some embodiments, the coding sequence encodes a BAG3 protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NQ:100. In some embodiments, the coding sequence encodes a BAG3 protein comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NQ:100. In some embodiments, the coding sequence encodes a BAG3 protein comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NQ:100. In some embodiments, the coding sequence encodes a BAG3 protein comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NQ:100. In some embodiments, the coding sequence encodes a BAG3 protein comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NQ:100. In some embodiments, the coding sequence encodes a BAG3 protein comprising an amino acid sequence having 100% sequence identity to SEQ ID NQ:100.

[0120] In some embodiments, the coding sequence encoding the BAG3 protein has at least 80% sequence identity to SEQ ID NQ:101. In some embodiments, the coding sequence encoding the BAG3 protein has at least 85% sequence identity to SEQ ID NQ:101. In some embodiments, the coding sequence encoding the BAG3 protein has at least 90% sequence identity to SEQ ID NQ:101 . In some embodiments, the coding sequence encoding the BAG3 protein has at least 95% sequence identity to SEQ ID NQ:101. In some embodiments, the coding sequence encoding the BAG3 protein has at least 96% sequence identity to SEQ ID NQ:101 . In some embodiments, the coding sequence encoding the BAG3 protein has at least 97% sequence identity to SEQ ID NO:101. In some embodiments, the coding sequence encoding the BAG3 protein has at least 98% sequence identity to SEQ ID NO:101 . In some embodiments, the coding sequence encoding the BAG3 protein has at least 99% sequence identity to SEQ ID NO:101 . In some embodiments, the coding sequence encoding the BAG3 protein has 100% sequence identity to SEQ ID NQ:101 .

[0121] In some embodiments, the coding sequence encoding the BAG3 protein has at least 80% sequence identity to SEQ ID NQ:102. In some embodiments, the coding sequence encoding the BAG3 protein has at least 85% sequence identity to SEQ ID NQ:102. In some embodiments, the coding sequence encoding the BAG3 protein has at least 90% sequence identity to SEQ ID NQ:102. In some embodiments, the coding sequence encoding the BAG3 protein has at least 95% sequence identity to SEQ ID NQ:102. In some embodiments, the coding sequence encoding the BAG3 protein has at least 96% sequence identity to SEQ ID NQ:102. In some embodiments, the coding sequence encoding the BAG3 protein has at least 97% sequence identity to SEQ ID NQ:102. In some embodiments, the coding sequence encoding the BAG3 protein has at least 98% sequence identity to SEQ ID NQ:102. In some embodiments, the coding sequence encoding the BAG3 protein has at least 99% sequence identity to SEQ ID NQ:102. In some embodiments, the coding sequence encoding the BAG3 protein has 100% sequence identity to SEQ ID NQ:102.

[0122] In some embodiments, the coding sequence encodes a BAG3 protein comprising a C151 R substitution, e.g., as set forth in SEQ ID NQ:150. In some embodiments, the coding sequence encodes a BAG3 protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NQ:150. In some embodiments, the coding sequence encodes a BAG3 protein comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NQ:150. In some embodiments, the coding sequence encodes a BAG3 protein comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NQ:150. In some embodiments, the coding sequence encodes a BAG3 protein comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NQ:150. In some embodiments, the coding sequence encodes a BAG3 protein comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NQ:150. In some embodiments, the coding sequence encodes a BAG3 protein comprising an amino acid sequence having 100% sequence identity to SEQ ID NQ:150.

[0123] In some embodiments, the coding sequence encoding the BAG3 protein has at least 80% sequence identity to SEQ ID NO:151. In some embodiments, the coding sequence encoding the BAG3 protein has at least 85% sequence identity to SEQ ID NO:151. In some embodiments, the coding sequence encoding the BAG3 protein has at least 90% sequence identity to SEQ ID NO:151 . In some embodiments, the coding sequence encoding the BAG3 protein has at least 95% sequence identity to SEQ ID NO:151. In some embodiments, the coding sequence encoding the BAG3 protein has at least 96% sequence identity to SEQ ID NO:151 . In some embodiments, the coding sequence encoding the BAG3 protein has at least 97% sequence identity to SEQ ID NO:151. In some embodiments, the coding sequence encoding the BAG3 protein has at least 98% sequence identity to SEQ ID NO:151 . In some embodiments, the coding sequence encoding the BAG3 protein has at least 99% sequence identity to SEQ ID NO:151 . In some embodiments, the coding sequence encoding the BAG3 protein has 100% sequence identity to SEQ ID NO:151 .

[0124] The MYBPC3 protein is preferably a human MYBPC3 protein, for example having an amino acid sequence of UniProt accession no. Q14896-1 or Q14896-2, set forth herein as SEQ ID NO:200 and SEQ ID NO:201 , respectively. Further exemplary MYBPC3 amino acid sequences are described in WO 2021/163357, the contents of which are incorporated herein by reference in their entirety.

[0125] In some embodiments, the coding sequence encodes a MYBPC3 protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID N0:200. In some embodiments, the coding sequence encodes a MYBPC3 protein comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NQ:200. In some embodiments, the coding sequence encodes a MYBPC3 protein comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NQ:200. In some embodiments, the coding sequence encodes a MYBPC3 protein comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NQ:200. In some embodiments, the coding sequence encodes a MYBPC3 protein comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NQ:200. In some embodiments, the coding sequence encodes a MYBPC3 protein comprising an amino acid sequence having 100% sequence identity to SEQ ID NQ:200.

[0126] In some embodiments, the coding sequence encodes a MYBPC3 protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NQ:201 . In some embodiments, the coding sequence encodes a MYBPC3 protein comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NQ:201 . In some embodiments, the coding sequence encodes a MYBPC3 protein comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NQ:201 . In some embodiments, the coding sequence encodes a MYBPC3 protein comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NQ:201 . In some embodiments, the coding sequence encodes a MYBPC3 protein comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NQ:201 . In some embodiments, the coding sequence encodes a MYBPC3 protein comprising an amino acid sequence having 100% sequence identity to SEQ ID NQ:201 .

[0127] In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 80% sequence identity to SEQ ID NQ:202. In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 85% sequence identity to SEQ ID NQ:202. In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 90% sequence identity to SEQ ID NQ:202. In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 95% sequence identity to SEQ ID NQ:202. In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 96% sequence identity to SEQ ID NQ:202. In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 97% sequence identity to SEQ ID NQ:202. In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 98% sequence identity to SEQ ID NQ:202. In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 99% sequence identity to SEQ ID NQ:202. In some embodiments, the coding sequence encoding the MYBPC3 protein has 100% sequence identity to SEQ ID NQ:202. [0128] In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 80% sequence identity to SEQ ID NO:203. In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 85% sequence identity to SEQ ID NO:203. In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 90% sequence identity to SEQ ID NQ:203. In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 95% sequence identity to SEQ ID NQ:203. In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 96% sequence identity to SEQ ID NQ:203. In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 97% sequence identity to SEQ ID NQ:203. In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 98% sequence identity to SEQ ID NQ:203. In some embodiments, the coding sequence encoding the MYBPC3 protein has at least 99% sequence identity to SEQ ID NQ:203. In some embodiments, the coding sequence encoding the MYBPC3 protein has 100% sequence identity to SEQ ID NQ:203.

[0129] The coding sequence encoding a BAG3 or MYBPC3 protein can be operably linked to one or more expression regulatory elements (EREs). The ERE(s) will generally be appropriate for a cell to be transduced with the BAG3 or MYBPC3 coding sequence. Numerous types of regulatory sequence and are known the art and may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences.

[0130] A promoter can be, for example, a muscle-specific promoter. Exemplary muscle specific promoters include myosin light chain 2 (MLC-2), BAG3, CK8e, MSEC-725a, CK7, alpha-myosin heavy chain (aMHC), desmin, creatine kinase (MCK), and cardiac Troponin T (cTnT or TNNT2) promoters. Further exemplary muscle specific promoters include MHCK7, myosin heavy polypeptide 7, cardiac muscle, beta (MYH7), Cysteine and glycine-rich protein 3 (CSRP3), histidine-rich calcium binding protein (HRC), and Myozenin-2 (MYOZ2).

[0131] In some embodiments, the promoter is an MLC-2 promoter, e.g., comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:300. In some embodiments, the promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:300. In some embodiments, the promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:300. In some embodiments, the promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:300. In some embodiments, the promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:300. In some embodiments, the promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:300. In some embodiments, the promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:300. In some embodiments, the promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:300.

[0132] In some embodiments, the promoter is a BAG3 promoter, e.g., comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:301. In some embodiments, the promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:301 . In some embodiments, the promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:301 . In some embodiments, the promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:301 . In some embodiments, the promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:301. In some embodiments, the promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:301 . In some embodiments, the promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:301 . In some embodiments, the promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:301.

[0133] In some embodiments, the promoter is a CK8e promoter, e.g., comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:302. In some embodiments, the promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:302. In some embodiments, the promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:302. In some embodiments, the promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:302. In some embodiments, the promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:302. In some embodiments, the promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:302. In some embodiments, the promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:302. In some embodiments, the promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:302.

[0134] In some embodiments, the promoter is a MSEC-725a promoter, e.g., comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:303. In some embodiments, the promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:303. In some embodiments, the promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:303. In some embodiments, the promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:303. In some embodiments, the promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:303. In some embodiments, the promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:303. In some embodiments, the promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:303. In some embodiments, the promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:303.

[0135] In some embodiments, the promoter is a CK7 promoter, e.g., comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:304. In some embodiments, the promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:304. In some embodiments, the promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:304. In some embodiments, the promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NO:304. In some embodiments, the promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:304. In some embodiments, the promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:304. In some embodiments, the promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:304. In some embodiments, the promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:304.

[0136] In some embodiments, the promoter is a MHCK7 promoter, e.g., comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:305. In some embodiments, the promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:305. In some embodiments, the promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:305. In some embodiments, the promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:305. In some embodiments, the promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:305. In some embodiments, the promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:305. In some embodiments, the promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:305. In some embodiments, the promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:305.

[0137] In some embodiments, the promoter is a MYH7 promoter, e.g., comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:306. In some embodiments, the promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:306. In some embodiments, the promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:306. In some embodiments, the promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:306. In some embodiments, the promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:306. In some embodiments, the promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:306. In some embodiments, the promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:306. In some embodiments, the promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:306.

[0138] In some embodiments, the promoter is a CSRP3 promoter, e.g., comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:307. In some embodiments, the promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:307. In some embodiments, the promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:307. In some embodiments, the promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NO:307. In some embodiments, the promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NO:307. In some embodiments, the promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:307. In some embodiments, the promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:307. In some embodiments, the promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:307.

[0139] In some embodiments, the promoter is a HRC promoter, e.g., comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:308. In some embodiments, the promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:308. In some embodiments, the promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:308. In some embodiments, the promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:308. In some embodiments, the promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:308. In some embodiments, the promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:308. In some embodiments, the promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:308. In some embodiments, the promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:308.

[0140] In some embodiments, the promoter is a MYOZ2 promoter, e.g., comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:309. In some embodiments, the promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:309. In some embodiments, the promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:309. In some embodiments, the promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:309. In some embodiments, the promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:309. In some embodiments, the promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:309. In some embodiments, the promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:309. In some embodiments, the promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:309.

[0141] Exemplary TNNT2 promoters are described in WO 2021/163357 (e.g., TNNT2-p600, TNNT2- p500, TNNT2-p400, and TNNT2-p300), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the promoter is a TNNT2 promoter, e.g., comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:310. In some embodiments, the promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:310. In some embodiments, the promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:310. In some embodiments, the promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NO:310. In some embodiments, the promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NO:310. In some embodiments, the promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:310. In some embodiments, the promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:310. In some embodiments, the promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:310. In some embodiments, the promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:311 . In some embodiments, the promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:311 . In some embodiments, the promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:311 . In some embodiments, the promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NO:311 . In some embodiments, the promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NO:311 . In some embodiments, the promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NO:311. In some embodiments, the promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NO:311 . In some embodiments, the promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO:311 .

[0142] In some embodiments, the promoter is a aMHC promoter, e.g., comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:312. In some embodiments, the promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:312. In some embodiments, the promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:312. In some embodiments, the promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NO:312. In some embodiments, the promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NO:312. In some embodiments, the promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NO:312. In some embodiments, the promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NO:312. In some embodiments, the promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO:312.

[0143] In some embodiments, the one or more expression regulatory elements include a posttranscriptional regulatory element sequence, for example an OPRE comprising a nucleotide sequence of SEQ ID NQ:400, or comprising a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:400. In some embodiments, the posttranscriptional regulatory element sequence comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:400. In some embodiments, the posttranscriptional regulatory element sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:400. In some embodiments, the posttranscriptional regulatory element sequence comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:400. In some embodiments, the posttranscriptional regulatory element sequence comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:400. In some embodiments, the posttranscriptional regulatory element sequence comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID N0:400. In some embodiments, the posttranscriptional regulatory element sequence comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID N0:400. In some embodiments, the posttranscriptional regulatory element sequence comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:400.

[0144] The polynucleotide can further comprise a polyadenylation signal sequence, for example a bovine growth hormone (BGH) polyadenylation signal sequence or a synthetic polyadenylation signal sequence. In some embodiments, a polyadenylation signal sequence comprises a nucleotide sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NQ:401 . In some embodiments, a polyadenylation signal sequence comprises a nucleotide sequence at least 95% identical to SEQ ID NQ:401. In some embodiments, a polyadenylation signal sequence comprises a nucleotide sequence at least 96% identical to SEQ ID NQ:401 . In some embodiments, a polyadenylation signal sequence comprises a nucleotide sequence at least 97% identical to SEQ ID NQ:401 . In some embodiments, a polyadenylation signal sequence comprises a nucleotide sequence at least 98% identical to SEQ ID NQ:401. In some embodiments, a polyadenylation signal sequence comprises a nucleotide sequence at least 99% identical to SEQ ID NQ:401 . In some embodiments, a polyadenylation signal sequence comprises a nucleotide sequence 100% identical to SEQ ID NQ:401 .

[0145] In some embodiments, a polyadenylation signal sequence comprises a nucleotide sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NQ:402. In some embodiments, a polyadenylation signal sequence comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 25. In some embodiments, a polyadenylation signal sequence comprises a nucleotide sequence at least 96% identical to SEQ ID NO: 25. In some embodiments, a polyadenylation signal sequence comprises a nucleotide sequence at least 97% identical to SEQ ID NO: 25. In some embodiments, a polyadenylation signal sequence comprises a nucleotide sequence at least 98% identical to SEQ ID NO:402. In some embodiments, a polyadenylation signal sequence comprises a nucleotide sequence at least 99% identical to SEQ ID NO:402. In some embodiments, a polyadenylation signal sequence comprises a nucleotide sequence 100% identical to SEQ ID NO:402.

[0146] The polynucleotide can further comprise, for example, when containing a BAG3 promoter, a BAG3 5’ UTR sequence and/or a BAG3 3’ UTR sequence. Exemplary BAG3 5’ UTR sequences are set forth in SEQ ID NQs:500-501 and an exemplary BAG3 3’ UTR sequence is set forth in SEQ ID NQ:502.ln some embodiments, a polynucleotide of the disclosure comprises a nucleotide sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NQ:500 or SEQ ID NQ:501 and/or comprises a nucleotide sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NQ:502.

[0147] The polynucleotide typically comprises AAV-derived inverted terminal repeat sequences (ITRs). In some embodiments, the ITRs are derived from AAV serotype 2. In some embodiments, the rAAV comprises a first ITR having at least 90% sequence identity to SEQ ID NQ:600, SEQ ID NQ:601 , SEQ ID NQ:602, or SEQ ID NQ:603 and a second ITR having at least 90% sequence identity to SEQ ID NQ:604 or SEQ ID NO:605. In some embodiments, the first ITR has at least 95% sequence identity to SEQ ID NQ:600, SEQ ID NO:601 , SEQ ID NQ:602, or SEQ ID NQ:603 and the second ITR has at least 95% sequence identity to SEQ ID NQ:604 or SEQ ID NQ:605. In some embodiments, the first ITR has at least 98% sequence identity to SEQ ID NQ:600, SEQ ID NQ:601 , SEQ ID NQ:602, or SEQ ID NQ:603 and the second ITR has at least 98% sequence identity to SEQ ID NQ:604 or SEQ ID NQ:605. In some embodiments, the first ITR has at least 99% sequence identity to SEQ ID NQ:600, SEQ ID NQ:601 , SEQ ID NQ:602, or SEQ ID NQ:603 and the second ITR has at least 99% sequence identity to SEQ ID NQ:604 or SEQ ID NQ:605. In some embodiments, the first ITR has 100% sequence identity to SEQ ID NQ:600, SEQ ID NQ:601 , SEQ ID NQ:602, or SEQ ID NQ:603 and the second ITR has 100% sequence identity to SEQ ID NQ:604 or SEQ ID NQ:605.

[0148] In some embodiments, the rAAV comprises a first ITR having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:600, SEQ ID NQ:601 , SEQ ID NQ:602, or SEQ ID NQ:603 and a second ITR having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:604 SEQ ID NQ:605, SEQ ID NQ:606, SEQ ID NQ:607, SEQ ID NQ:608, SEQ ID NQ:609, or SEQ ID NQ:610.

[0149] In some embodiments, the rAAV comprises a first ITR having 100% sequence identity to SEQ ID NQ:600 and a second ITR having 100% sequence identity to SEQ ID NQ:607. In some embodiments, the rAAV comprises a first ITR having 100% sequence identity to SEQ ID NQ:600 and a second ITR having 100% sequence identity to SEQ ID NQ:608.

[0150] Exemplary polynucleotides that can be included in rAAV of the disclosure are set forth in SEQ ID NQS:800-810 and SEQ ID NQS:900-910. In some embodiments, a rAAV of the disclosure comprises a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NQ:810. In some embodiments, a rAAV of the disclosure comprises a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NQ:910.

6.3. Methods of Producing rAAV

[0151] The rAAV of the disclosure comprise a BAG3 or MYBPC3 coding sequence. The coding sequence and any ERE can replace the AAV genomic coding region (e.g., replace the AAV rep and cap genes). The transgene coding sequence and ERE are generally flanked on either side by AAV inverted terminal repeat (ITR) regions, although a single ITR may be sufficient to carry out the functions normally associated with configurations comprising two ITRs (see, for example, WO 94/13788), and vector constructs with only one ITR can thus be employed in conjunction with the rAAV of the present disclosure.

[0152] In order to replicate and package the vector, the missing functions are complemented with a packaging gene, or a plurality thereof, which together encode the necessary functions for the various missing rep and/or cap gene products. The packaging genes or gene cassettes are in one embodiment not flanked by AAV ITRs and in one embodiment do not share any substantial homology with the rAAV genome. [0153] The rAAV vector construct, and the complementary packaging gene constructs can be implemented in a number of different forms. Viral particles, plasmids, and stably transformed host cells can all be used to introduce such constructs into the packaging cell, either transiently or stably.

[0154] In certain embodiments, the AAV vector and complementary packaging gene(s), if any, are provided in the form of bacterial plasmids, AAV particles, or any combination thereof. In other embodiments, either the AAV vector sequence, the packaging gene(s), or both, are provided in the form of genetically altered (preferably inheritably altered) eukaryotic cells. The development of host cells inheritably altered to express the AAV vector sequence, AAV packaging genes, or both, provides an established source of the material that is expressed at a reliable level.

[0155] A variety of different genetically altered cells can thus be used in the context of this disclosure. By way of illustration, a mammalian host cell may be used with at least one intact copy of a stably integrated rAAV vector. An AAV packaging plasmid comprising at least an AAV rep gene operably linked to a promoter can be used to supply replication functions (as described in U.S. Pat. No. 5,658,776). Alternatively, a stable mammalian cell line with an AAV rep gene operably linked to a promoter can be used to supply replication functions (see, e.g., WO 95/13392; WO 98/23018; and U.S. Patent No. 5,656,785). The AAV cap gene, providing the encapsidation proteins as described above, can be provided together with an AAV rep gene or separately (see, e.g., the above- referenced patent documents as well as WO 98/27204.

[0156] Thus, the rAAV of the disclosure can be assembled by, for example, expression of its components in a packaging host cell. The components of a virus particle (e.g., rep sequences, cap sequences, inverted terminal repeat (ITR) sequences) can be introduced into a packaging host cell using one or more viral vectors.

[0157] Once assembled, rAAV particles can be purified, if desired, using routine methods. As used herein, “purified” virus particles refer to virus particles that are removed from components in the mixture in which they were made such as, but not limited to, viral components (e.g., rep sequences, cap sequences), packaging host cells, and partially- or incompletely- assembled virus particles.

6.4. Pharmaceutical Compositions

[0158] In one aspect, the present disclosure provides a pharmaceutical composition comprising the rAAV of the present disclosure and a pharmaceutically acceptable carrier.

[0159] The pharmaceutical composition can be used to deliver the rAAV to a mammalian subject (e.g., human subject) in need of BAG3 or MYBPC3 gene expression, e.g., a subject suffering from or at risk of a cardiomyopathy as described herein, for example a subject having a BAG3 and/or MYBPC3 mutation. When the pharmaceutical composition is administered, the rAAV can achieve an improved therapeutic index through high levels of transgene protein expression and/or by a higher infection of cardiac cells and/or reduced expression of liver cells per viral genome administered as compared to a control rAAV which includes an unmodified capsid protein, e.g., a capsid protein lacking a targeting peptide and/or liver toggle mutation when the control rAAV is administered by the same route of administration and in the same dose.

[0160] The pharmaceutical composition can be formulated using one or more carriers, excipients, stabilizers and adjuvants to, for example: (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release; (4) alter the biodistribution (e.g., target the rAAV particle to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein in vivo.

[0161] Formulations of the pharmaceutical compositions provided herein can include, without limitation, saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, water, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, nanoparticle mimics and combinations thereof.

[0162] Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the active ingredient with a carrier and/or one or more other accessory ingredients (e.g., excipients, stabilizers and adjuvants).

[0163] A pharmaceutical composition in accordance with the present disclosure can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a unit dose refers to a discrete amount of the pharmaceutical composition including a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0164] Relative amounts of the active ingredient (e.g., rAAV), the pharmaceutically acceptable carrier, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure can vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.

[0165] Various carriers, excipients, stabilizers and adjuvants for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 22nd Revised Ed., Pharmaceutical Press, 2012; incorporated herein by reference in its entirety). The use of suitable conventional carriers, excipients, stabilizers and adjuvants is contemplated within the scope of the present disclosure.

[0166] In some embodiments, the pharmaceutical composition is in the form of a solution containing concentrations of from about 1 x 10¹ to about 1 x 10¹⁶ genome copies (GCs)/ml of rAAV (e.g., a solution containing concentrations of from about 1 x 10³ to about 1 x 10¹⁴ GCs/ml.

6.5. Methods of Treatment [0167] The rAAVs or pharmaceutical compositions described are useful for delivering a polynucleotide comprising a BAG3 protein or MYBCP3 protein coding sequence to the heart of subjects (preferably human subjects), for example subjects suffering from or at risk of a cardiac disease. In some embodiments, the subject has a cardiac disease. In other embodiments, the subject is at risk of a cardiac disease, e.g., due to mutation in the subject’s BAG3 or MYBPC3.

[0168] The rAAVs or pharmaceutical compositions described are useful in the treatment of subjects (preferably human subjects) suffering from or at risk of a cardiac disease and/or carrying mutations in the BAG3 or MYBPC3 gene. Exemplary cardiac diseases include genetic cardiomyopathies, dilated cardiomyopathy (DCM) (e.g., idiopathic DCM, familial DCM, or non-familial DCM), hypertrophic cardiomyopathy, non-ischemic cardiomyopathy, heart failure (e.g., congestive heart failure, heart failure due to reduced ejection fraction, heart failure due to coronary artery disease, acute heart failure, or endstage heart failure), restrictive cardiomyopathy, left-ventricular non-compaction, atherosclerosis, coronary artery disease, ischemic heart disease, myocarditis, hypertensive heart disease, valvular disease, congenital heart disease, or myocardial infarction.

[0169] In some embodiments, the subject has or is at risk of a genetic cardiomyopathy (e.g., a subject having a BAG3 or MYBPC3 mutation).

[0170] In some embodiments, the subject (e.g., a subject having a BAG3 mutation or MYBPC3 mutation) has or is at risk of dilated cardiomyopathy (DCM). In some embodiments, the DCM is idiopathic DCM. In other embodiments, the DCM is familial DCM. In other embodiments, the DCM is non-familial DCM. In some embodiments, the subject having DCM or at risk of DCM has a BAG3 mutation.

[0171] In some embodiments, the subject (e.g., a subject having a BAG3 mutation) has or is at risk of non-ischemic cardiomyopathy.

[0172] In some embodiments, the subject (e.g., a subject having a BAG3 mutation) has or is at risk of heart failure. In some embodiments, the heart failure is due to reduced ejection fraction. In some embodiments, the heart failure is due to coronary artery disease.

[0173] In some embodiments, the subject (e.g., a subject having a MYBPC3 mutation) has or is at risk of hypertrophic cardiomyopathy (HCM).

[0174] In some embodiments, the subject (e.g., a subject having a MYBPC3 mutation) has or is at risk of dilated cardiomyopathy.

[0175] In some embodiments, the subject (e.g., a subject having a MYBPC3 mutation) has or is at risk of restrictive cardiomyopathy.

[0176] In some embodiments, the subject (e.g., a subject having a MYBPC3 mutation) has or is at risk of left-ventricular non-compaction. [0177] The rAAV of the disclosure are typically administered in sufficient amounts to transduce or infect the desired cells and to provide sufficient levels of gene transfer and expression to provide a therapeutic benefit to subjects suffering from or at risk of the cardiac disease and/or carrying a mutation in the BAG3 or MYBPC3 gene, without undue adverse effects.

[0178] A rAAV of the present disclosure can be administered to a subject in a suitable pharmaceutical carrier, e.g., as described in Section 6.4.

[0179] Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the heart, orally, intranasally, intratracheally, intrathecally, intravenously, intramuscularly, intraocularly, subcutaneously, intradermally, or by other routes of administration. Routes of administration can be combined, if desired.

[0180] The dose of rAAV administered to a subject will depend primarily on factors such as the age, weight, and health (e.g., disease progression) of the subject. For example, a therapeutically effective dosage of a viral vector to be administered to a human subject generally is in the range of from about 0.1 ml to about 10 ml of a solution containing concentrations of from about 1 x 10¹ to about 1 x 10¹⁶ genome copies (GCs)Zml of viruses (e.g., a solution containing concentrations of from about 1 x 10³ to about 1 x 10¹⁴ GCs/ml). In some embodiments, the total dose of the rAAV administered to a subject is less than 3 x 10¹⁴ GCs, e.g., 1 x 10¹⁴ GCs or less, 5 x 10¹³ GCs or less, 1 x 10¹³ GCs or less, 5 x 10¹² GCs or less, or 1 x 10¹² GCs or less.

[0181] In some embodiments, a therapeutically effective dosage of a rAAV administered to a human subject is about 1 e12 vg/kg to 1 e14 vg/kg. In some embodiments, a therapeutically effective dosage of a rAAV to be administered to a human subject is less than 1 e14 vg/kg. In some embodiments, a therapeutically effective dosage of a rAAV to be administered to a human subject is 1 ,5e12 vg/kg to 1.5e13 vg/kg. In some embodiments, a therapeutically effective dosage of a rAAV to be administered to a human subject is 1 e13 vg/kg. In some embodiments, a therapeutically effective dosage of a rAAV to be administered to a human subject is 1 e13 vg/kg to 1 e14 vg/kg. In some embodiments, a therapeutically effective dosage of a rAAV to be administered to a human subject is 3e13 vg/kg.

[0182] Transduction and/or expression of the BAG3 or MYBPC3 transgene can be monitored at various time points following administration by DNA, RNA, or protein assays.

7. SPECIFIC EMBODIMENTS

[0183] The present disclosure is exemplified by the specific embodiments below.

1 . A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a modified capsid protein having a targeting peptide within variable region VIII (VR VIII) comprising the amino acid sequence X1X2X3RGDX7X8X9X10, where X1X2X3 is SAQ, ASS, ENK, or ENR and X7, Xs, X9, and X10 are independently selected from any amino acid residue; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein or a myosin-binding protein C, cardiac-type (MYBPC3) protein.

2. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a modified capsid protein having a targeting peptide within variable region VIII (VR VIII) comprising the amino acid sequence X1X2X3RGDRGQI (SEQ ID NO:12), X1X2X3RGDFNNL (SEQ ID NO:13), or XiX₂X₃RGDYTSM (SEQ ID NO:14), where Xi, X₂, and X3 are independently selected from any amino acid residue; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein or a myosin-binding protein C, cardiac-type (MYBPC3) protein.

3. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a modified capsid protein having a targeting peptide within variable region VIII (VR VIII), wherein the targeting peptide comprises the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein or a myosin-binding protein C, cardiac-type (MYBPC3) protein.

4. The rAAV of any one of embodiments 1 to 3, wherein the targeting peptide is inserted between amino acid positions corresponding to positions 585 and 589 of an AAV9 reference sequence as set forth in SEQ ID NO:32 such that amino acids corresponding to positions 586, 587 and 588 of the reference sequence are replaced with the amino acids of the targeting peptide.

5. The rAAV of any one of embodiments 1 to 4, wherein the modified capsid protein comprises at least one liver-toggle mutation as compared to a reference capsid protein.

6. The rAAV of embodiment 5, wherein the at least one liver-toggle mutation comprises an alanine (A) at an amino acid position corresponding to position 267 in an AAV9 reference sequence as set forth in SEQ ID NO:32.

7. The rAAV of embodiment 5 or embodiment 6, wherein the at least one liver-toggle mutation comprises a threonine (T) at an amino acid position corresponding to position 269 in an AAV9 reference sequence as set forth in SEQ ID NO:32.

8. The rAAV of any one of embodiments 1 to 4, wherein the modified capsid protein comprises a serine (S) at an amino acid position corresponding to position 262 in an AAV9 reference sequence as set forth in SEQ ID NO:32, a glycine (G) at an amino acid position corresponding to position 263 in an AAV9 reference sequence as set forth in SEQ ID NO:32, a threonine (T) at an amino acid position corresponding to position 265 in an AAV9 reference sequence as set forth in SEQ ID NO:32, and a threonine at an amino acid position corresponding to position 273 in an AAV9 reference sequence as set forth in SEQ ID NO:32.

9. The rAAV of any one of embodiments 1 to 4, wherein the modified capsid protein comprises a peptide segment within variable region I (VR I) comprising an amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

10. The rAAV of embodiment 9, wherein the peptide segment comprises the amino acid sequence of SEQ ID NO:5.

11 . The rAAV of embodiment 9, wherein the peptide segment comprises the amino acid sequence of SEQ ID NO:6.

12. The rAAV of embodiment 9, wherein the peptide segment comprises the amino acid sequence of SEQ ID NOT.

13. The rAAV of any one of embodiments 9 to 12, wherein the amino acids of the peptide segment correspond to positions 262 to 273 of an AAV9 reference sequence as set forth in SEQ ID NO:32.

14. The rAAV of any one of embodiments 1 and 4 to 13, when depending directly or indirectly from embodiment 1 , wherein X1X2X3 is SAQ.

15. The rAAV of any one of embodiments 2 and 4 to 13, when depending directly or indirectly from embodiment 2, wherein the targeting peptide comprises the amino acid sequence X1X2X3RGDRGQI (SEQ ID NO:12).

16. The rAAV of any one of embodiments 1 to 15, wherein the targeting peptide comprises the amino acid sequence of SEQ ID NO:1 .

17. The rAAV of any one of embodiments 14 to 16, wherein the capsid comprises a VP1 capsid protein having at least 90% sequence identity to SEQ ID NO:8.

18. The rAAV of any one of embodiments 14 to 16, wherein the capsid comprises a VP1 capsid protein having at least 95% sequence identity to SEQ ID NO:8.

19. The rAAV of any one of embodiments 14 to 16, wherein the capsid comprises a VP1 capsid protein having at least 96% sequence identity to SEQ ID NO:8.

20. The rAAV of any one of embodiments 14 to 16, wherein the capsid comprises a VP1 capsid protein having at least 97% sequence identity to SEQ ID NO:8.

21 . The rAAV of any one of embodiments 14 to 16, wherein the capsid comprises a VP1 capsid protein having at least 98% sequence identity to SEQ ID NO:8. 22. The rAAV of any one of embodiments 14 to 16, wherein the capsid comprises a VP1 capsid protein having at least 99% sequence identity to SEQ ID NO:8.

23. The rAAV of any one of embodiments 14 to 16, wherein the capsid comprises a VP1 capsid protein having 100% sequence identity to SEQ ID NO:8.

24. The rAAV of any one of embodiments 14 to 23, wherein the capsid comprises a VP2 capsid protein having at least 90% sequence identity to amino acids 138 to 743 of SEQ ID NO:8.

25. The rAAV of any one of embodiments 14 to 23, wherein the capsid comprises a VP2 capsid protein having at least 95% sequence identity to amino acids 138 to 743 of SEQ ID NO:8.

26. The rAAV of any one of embodiments 14 to 23, wherein the capsid comprises a VP2 capsid protein having at least 96% sequence identity to amino acids 138 to 743 of SEQ ID NO:8.

27. The rAAV of any one of embodiments 14 to 23, wherein the capsid comprises a VP2 capsid protein having at least 97% sequence identity to amino acids 138 to 743 of SEQ ID NO:8.

28. The rAAV of any one of embodiments 14 to 23, wherein the capsid comprises a VP2 capsid protein having at least 98% sequence identity to amino acids 138 to 743 of SEQ ID NO:8.

29. The rAAV of any one of embodiments 14 to 23, wherein the capsid comprises a VP2 capsid protein having at least 99% sequence identity to amino acids 138 to 743 of SEQ ID NO:8.

30. The rAAV of any one of embodiments 14 to 23, wherein the capsid comprises a VP2 capsid protein having 100% sequence identity to amino acids 138 to 743 of SEQ ID NO:8.

31 . The rAAV of any one of embodiments 14 to 30, wherein the capsid comprises a VP3 capsid protein having at least 90% sequence identity to amino acids 203 to 743 of SEQ ID NO:8.

32. The rAAV of any one of embodiments 14 to 30, wherein the capsid comprises a VP3 capsid protein having at least 95% sequence identity to amino acids 203 to 743 of SEQ ID NO:8.

33. The rAAV of any one of embodiments 14 to 30, wherein the capsid comprises a VP3 capsid protein having at least 96% sequence identity to amino acids 203 to 743 of SEQ ID NO:8.

34. The rAAV of any one of embodiments 14 to 30, wherein the capsid comprises a VP3 capsid protein having at least 97% sequence identity to amino acids 203 to 743 of SEQ ID NO:8.

35. The rAAV of any one of embodiments 14 to 30, wherein the capsid comprises a VP3 capsid protein having at least 98% sequence identity to amino acids 203 to 743 of SEQ ID NO:8.

36. The rAAV of any one of embodiments 14 to 30, wherein the capsid comprises a VP3 capsid protein having at least 99% sequence identity to amino acids 203 to 743 of SEQ ID NO:8. 37. The rAAV of any one of embodiments 14 to 30, wherein the capsid comprises a VP3 capsid protein having 100% sequence identity to amino acids 203 to 743 of SEQ ID NO:8.

38. The rAAV of any one of embodiments 1 and 4 to 13, when depending directly or indirectly from embodiment 1 , wherein X1X2X3 is ASS.

39. The rAAV of any one of embodiments 2 and 4 to 13, when depending directly or indirectly from embodiment 2, wherein the targeting peptide comprises the amino acid sequence X1X2X3RGDFNNL (SEQ ID NO:13).

40. The rAAV of any one of embodiments 1 to 13 and 38 to 39, wherein the targeting peptide comprises the amino acid sequence of SEQ ID NO:2.

41 . The rAAV of any one of embodiments 38 to 40, wherein the capsid comprises a VP1 capsid protein having at least 90% sequence identity to SEQ ID NO:9.

42. The rAAV of any one of embodiments 38 to 40, wherein the capsid comprises a VP1 capsid protein having at least 95% sequence identity to SEQ ID NO:9.

43. The rAAV of any one of embodiments 38 to 40, wherein the capsid comprises a VP1 capsid protein having at least 96% sequence identity to SEQ ID NO:9.

44. The rAAV of any one of embodiments 38 to 40, wherein the capsid comprises a VP1 capsid protein having at least 97% sequence identity to SEQ ID NO:9.

45. The rAAV of any one of embodiments 38 to 40, wherein the capsid comprises a VP1 capsid protein having at least 98% sequence identity to SEQ ID NO:9.

46. The rAAV of any one of embodiments 38 to 40, wherein the capsid comprises a VP1 capsid protein having at least 99% sequence identity to SEQ ID NO:9.

47. The rAAV of any one of embodiments 38 to 40, wherein the capsid comprises a VP1 capsid protein having 100% sequence identity to SEQ ID NO:9.

48. The rAAV of any one of embodiments 38 to 47, wherein the capsid comprises a VP2 capsid protein having at least 90% sequence identity to amino acids 138 to 743 of SEQ ID NO:9.

49. The rAAV of any one of embodiments 38 to 47, wherein the capsid comprises a VP2 capsid protein having at least 95% sequence identity to amino acids 138 to 743 of SEQ ID NO:9.

50. The rAAV of any one of embodiments 38 to 47, wherein the capsid comprises a VP2 capsid protein having at least 96% sequence identity to amino acids 138 to 743 of SEQ ID NO:9.

51 . The rAAV of any one of embodiments 38 to 47, wherein the capsid comprises a VP2 capsid protein having at least 97% sequence identity to amino acids 138 to 743 of SEQ ID NO:9. 52. The rAAV of any one of embodiments 38 to 47, wherein the capsid comprises a VP2 capsid protein having at least 98% sequence identity to amino acids 138 to 743 of SEQ ID NO:9.

53. The rAAV of any one of embodiments 38 to 47, wherein the capsid comprises a VP2 capsid protein having at least 99% sequence identity to amino acids 138 to 743 of SEQ ID NO:9.

54. The rAAV of any one of embodiments 38 to 47, wherein the capsid comprises a VP2 capsid protein having 100% sequence identity to amino acids 138 to 743 of SEQ ID NO:9.

55. The rAAV of any one of embodiments 38 to 54, wherein the capsid comprises a VP3 capsid protein having at least 90% sequence identity to amino acids 203 to 743 of SEQ ID NO:9.

56. The rAAV of any one of embodiments 38 to 54, wherein the capsid comprises a VP3 capsid protein having at least 95% sequence identity to amino acids 203 to 743 of SEQ ID NO:9.

57. The rAAV of any one of embodiments 38 to 54, wherein the capsid comprises a VP3 capsid protein having at least 96% sequence identity to amino acids 203 to 743 of SEQ ID NO:9.

58. The rAAV of any one of embodiments 38 to 54, wherein the capsid comprises a VP3 capsid protein having at least 97% sequence identity to amino acids 203 to 743 of SEQ ID NO:9.

59. The rAAV of any one of embodiments 38 to 54, wherein the capsid comprises a VP3 capsid protein having at least 98% sequence identity to amino acids 203 to 743 of SEQ ID NO:9.

60. The rAAV of any one of embodiments 38 to 54, wherein the capsid comprises a VP3 capsid protein having at least 99% sequence identity to amino acids 203 to 743 of SEQ ID NO:9.

61 . The rAAV of any one of embodiments 38 to 54, wherein the capsid comprises a VP3 capsid protein having 100% sequence identity to amino acids 203 to 743 of SEQ ID NO:9.

62. The rAAV of any one of embodiments 1 and 4 to 13, when depending directly or indirectly from embodiment 1 , wherein X1X2X3 is ENK.

63. The rAAV of any one of embodiments 2 and 4 to 13, when depending directly or indirectly from embodiment 2, wherein the targeting peptide comprises the amino acid sequence X1X2X3RGDYTSM (SEQ ID NO:14).

64. The rAAV of any one of embodiments 1 to 13 and 62 to 63, wherein the targeting peptide comprises the amino acid sequence of SEQ ID NO:3.

65. The rAAV of any one of embodiments 62 to 64, wherein the capsid comprises a VP1 capsid protein having at least 90% sequence identity to SEQ ID NQ:10.

66. The rAAV of any one of embodiments 62 to 64, wherein the capsid comprises a VP1 capsid protein having at least 95% sequence identity to SEQ ID NQ:10. 67. The rAAV of any one of embodiments 62 to 64, wherein the capsid comprises a VP1 capsid protein having at least 96% sequence identity to SEQ ID NO:10.

68. The rAAV of any one of embodiments 62 to 64, wherein the capsid comprises a VP1 capsid protein having at least 97% sequence identity to SEQ ID NO:10.

69. The rAAV of any one of embodiments 62 to 64, wherein the capsid comprises a VP1 capsid protein having at least 98% sequence identity to SEQ ID NQ:10.

70. The rAAV of any one of embodiments 62 to 64, wherein the capsid comprises a VP1 capsid protein having at least 99% sequence identity to SEQ ID NQ:10.

71 . The rAAV of any one of embodiments 62 to 64, wherein the capsid comprises a VP1 capsid protein having 100% sequence identity to SEQ ID NQ:10.

72. The rAAV of any one of embodiments 62 to 71 , wherein the capsid comprises a VP2 capsid protein having at least 90% sequence identity to amino acids 138 to 743 of SEQ ID NQ:10.

73. The rAAV of any one of embodiments 62 to 71 , wherein the capsid comprises a VP2 capsid protein having at least 95% sequence identity to amino acids 138 to 743 of SEQ ID NQ:10.

74. The rAAV of any one of embodiments 62 to 71 , wherein the capsid comprises a VP2 capsid protein having at least 96% sequence identity to amino acids 138 to 743 of SEQ ID NQ:10.

75. The rAAV of any one of embodiments 62 to 71 , wherein the capsid comprises a VP2 capsid protein having at least 97% sequence identity to amino acids 138 to 743 of SEQ ID NQ:10.

76. The rAAV of any one of embodiments 62 to 71 , wherein the capsid comprises a VP2 capsid protein having at least 98% sequence identity to amino acids 138 to 743 of SEQ ID NQ:10.

77. The rAAV of any one of embodiments 62 to 71 , wherein the capsid comprises a VP2 capsid protein having at least 99% sequence identity to amino acids 138 to 743 of SEQ ID NQ:10.

78. The rAAV of any one of embodiments 62 to 71 , wherein the capsid comprises a VP2 capsid protein having 100% sequence identity to amino acids 138 to 743 of SEQ ID NQ:10.

79. The rAAV of any one of embodiments 62 to 78, wherein the capsid comprises a VP3 capsid protein having at least 90% sequence identity to amino acids 203 to 743 of SEQ ID NQ:10.

80. The rAAV of any one of embodiments 62 to 78, wherein the capsid comprises a VP3 capsid protein having at least 95% sequence identity to amino acids 203 to 743 of SEQ ID NQ:10.

81 . The rAAV of any one of embodiments 62 to 78, wherein the capsid comprises a VP3 capsid protein having at least 96% sequence identity to amino acids 203 to 743 of SEQ ID NQ:10. 82. The rAAV of any one of embodiments 62 to 78, wherein the capsid comprises a VP3 capsid protein having at least 97% sequence identity to amino acids 203 to 743 of SEQ ID NO:10.

83. The rAAV of any one of embodiments 62 to 78, wherein the capsid comprises a VP3 capsid protein having at least 98% sequence identity to amino acids 203 to 743 of SEQ ID NO:10.

84. The rAAV of any one of embodiments 62 to 78, wherein the capsid comprises a VP3 capsid protein having at least 99% sequence identity to amino acids 203 to 743 of SEQ ID NQ:10.

85. The rAAV of any one of embodiments 62 to 78, wherein the capsid comprises a VP3 capsid protein having 100% sequence identity to amino acids 203 to 743 of SEQ ID NQ:10.

86. The rAAV of any one of embodiments 1 and 4 to 13, when depending directly or indirectly from embodiment 1 , wherein X1X2X3 is ENR.

87. The rAAV of any one of embodiments 2 and 4 to 13, when depending directly or indirectly from embodiment 2, wherein the targeting peptide comprises the amino acid sequence X1X2X3RGDFNNL (SEQ ID NO:13).

88. The rAAV of any one of embodiments 1 to 13 and 86 to 87, wherein the targeting peptide comprises the amino acid sequence of SEQ ID NO:4.

89. The rAAV of any one of embodiments 86 to 88, wherein the capsid comprises a VP1 capsid protein having at least 90% sequence identity to SEQ ID NO:11 .

90. The rAAV of any one of embodiments 86 to 88, wherein the capsid comprises a VP1 capsid protein having at least 95% sequence identity to SEQ ID NO:11 .

91 . The rAAV of any one of embodiments 86 to 88, wherein the capsid comprises a VP1 capsid protein having at least 96% sequence identity to SEQ ID NO:11 .

92. The rAAV of any one of embodiments 86 to 88, wherein the capsid comprises a VP1 capsid protein having at least 97% sequence identity to SEQ ID NO:11 .

93. The rAAV of any one of embodiments 86 to 88, wherein the capsid comprises a VP1 capsid protein having at least 98% sequence identity to SEQ ID NO:11 .

94. The rAAV of any one of embodiments 86 to 88, wherein the capsid comprises a VP1 capsid protein having at least 99% sequence identity to SEQ ID NO:11 .

95. The rAAV of any one of embodiments 86 to 88, wherein the capsid comprises a VP1 capsid protein having 100% sequence identity to SEQ ID NO:11 .

96. The rAAV of any one of embodiments 86 to 95, wherein the capsid comprises a VP2 capsid protein having at least 90% sequence identity to amino acids 138 to 743 of SEQ ID NO:11. 97. The rAAV of any one of embodiments 86 to 95, wherein the capsid comprises a VP2 capsid protein having at least 95% sequence identity to amino acids 138 to 743 of SEQ ID NO:11 .

98. The rAAV of any one of embodiments 86 to 95, wherein the capsid comprises a VP2 capsid protein having at least 96% sequence identity to amino acids 138 to 743 of SEQ ID NO:11 .

99. The rAAV of any one of embodiments 86 to 95, wherein the capsid comprises a VP2 capsid protein having at least 97% sequence identity to amino acids 138 to 743 of SEQ ID NO:11 .

100. The rAAV of any one of embodiments 86 to 95, wherein the capsid comprises a VP2 capsid protein having at least 98% sequence identity to amino acids 138 to 743 of SEQ ID NO:11 .

101 . The rAAV of any one of embodiments 86 to 95, wherein the capsid comprises a VP2 capsid protein having at least 99% sequence identity to amino acids 138 to 743 of SEQ ID NO:11.

102. The rAAV of any one of embodiments 86 to 95, wherein the capsid comprises a VP2 capsid protein having 100% sequence identity to amino acids 138 to 743 of SEQ ID NO:11.

103. The rAAV of any one of embodiments 86 to 102, wherein the capsid comprises a VP3 capsid protein having at least 90% sequence identity to amino acids 203 to 743 of SEQ ID NO:11 .

104. The rAAV of any one of embodiments 86 to 102, wherein the capsid comprises a VP3 capsid protein having at least 95% sequence identity to amino acids 203 to 743 of SEQ ID NO:11 .

105. The rAAV of any one of embodiments 86 to 102, wherein the capsid comprises a VP3 capsid protein having at least 96% sequence identity to amino acids 203 to 743 of SEQ ID NO:11 .

106. The rAAV of any one of embodiments 86 to 102, wherein the capsid comprises a VP3 capsid protein having at least 97% sequence identity to amino acids 203 to 743 of SEQ ID NO:11 .

107. The rAAV of any one of embodiments 86 to 102, wherein the capsid comprises a VP3 capsid protein having at least 98% sequence identity to amino acids 203 to 743 of SEQ ID NO:11 .

108. The rAAV of any one of embodiments 86 to 102, wherein the capsid comprises a VP3 capsid protein having at least 99% sequence identity to amino acids 203 to 743 of SEQ ID NO:11 .

109. The rAAV of any one of embodiments 86 to 102, wherein the capsid comprises a VP3 capsid protein having 100% sequence identity to amino acids 203 to 743 of SEQ ID NO:11.

110. The rAAV of any one of embodiments 1 to 109, wherein the coding sequence is codon optimized for human cells.

111. The rAAV of any one of embodiments 1 to 109, wherein the coding sequence is a wildtype coding sequence. 112. The rAAV of any one of embodiments 1 to 111 , wherein the polynucleotide comprises a coding sequence encoding a BAG3 protein.

113. The rAAV of embodiment 112, wherein the BAG3 protein is a human BAG3 protein.

114. The rAAV of embodiment 112 or embodiment 113, wherein the BAG3 protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID N0:100.

115. The rAAV of embodiment 112 or embodiment 113, wherein the BAG3 protein comprises an amino acid sequence having at least 96% sequence identity to SEQ ID N0:100.

116. The rAAV of embodiment 112 or embodiment 113, wherein the BAG3 protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NQ:100.

117. The rAAV of embodiment 112 or embodiment 113, wherein the BAG3 protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NQ:100.

118. The rAAV of embodiment 112 or embodiment 113, wherein the BAG3 protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NQ:100.

119. The rAAV of embodiment 112 or embodiment 113, wherein the BAG3 protein comprises an amino acid sequence having 100% sequence identity to SEQ ID NQ:100.

120. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 80% sequence identity to SEQ ID NQ:101 .

121 . The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 85% sequence identity to SEQ ID NQ:101 .

122. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 90% sequence identity to SEQ ID NQ:101 .

123. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 95% sequence identity to SEQ ID NQ:101 .

124. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 96% sequence identity to SEQ ID NQ:101 .

125. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 97% sequence identity to SEQ ID NQ:101 .

126. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 98% sequence identity to SEQ ID NQ:101 . 127. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 99% sequence identity to SEQ ID NO:101 .

128. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has 100% sequence identity to SEQ ID NO:101.

129. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 80% sequence identity to SEQ ID NQ:102.

130. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 85% sequence identity to SEQ ID NQ:102.

131 . The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 90% sequence identity to SEQ ID NQ:102.

132. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 95% sequence identity to SEQ ID NQ:102.

133. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 96% sequence identity to SEQ ID NQ:102.

134. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 97% sequence identity to SEQ ID NQ:102.

135. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 98% sequence identity to SEQ ID NQ:102.

136. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 99% sequence identity to SEQ ID NQ:102.

137. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has 100% sequence identity to SEQ ID NQ:102.

138. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 80% sequence identity to SEQ ID NQ:103.

139. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 85% sequence identity to SEQ ID NQ:103.

140. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 90% sequence identity to SEQ ID NQ:103.

141 . The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 95% sequence identity to SEQ ID NQ:103. 142. The rAAV of any one of embodiments 112 to 1 19, wherein the coding sequence encoding the BAG3 protein has at least 96% sequence identity to SEQ ID NO:103.

143. The rAAV of any one of embodiments 112 to 1 19, wherein the coding sequence encoding the BAG3 protein has at least 97% sequence identity to SEQ ID NO:103.

144. The rAAV of any one of embodiments 112 to 1 19, wherein the coding sequence encoding the BAG3 protein has at least 98% sequence identity to SEQ ID NQ:103.

145. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has at least 99% sequence identity to SEQ ID NQ:103.

146. The rAAV of any one of embodiments 112 to 119, wherein the coding sequence encoding the BAG3 protein has 100% sequence identity to SEQ ID NQ:103.

147. The rAAV of embodiment 112 or embodiment 113, wherein the BAG3 protein comprises a C151 R substitution.

148. The rAAV of embodiment 112, or embodiment 1 13, or embodiment 147, wherein the BAG3 protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NQ:150.

149. The rAAV of embodiment 112, or embodiment 1 13, or embodiment 147, wherein the BAG3 protein comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NQ:150.

150. The rAAV of embodiment 112, or embodiment 1 13, or embodiment 147, wherein the BAG3 protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NQ:150.

151 . The rAAV of embodiment 112, or embodiment 1 13, or embodiment 147, wherein the BAG3 protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NQ:150.

152. The rAAV of embodiment 112, or embodiment 1 13, or embodiment 147, wherein the BAG3 protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NQ:150.

153. The rAAV of embodiment 112, or embodiment 1 13, or embodiment 147, wherein the BAG3 protein comprises an amino acid sequence having 150% sequence identity to SEQ ID NO: 150.

154. The rAAV of any one of embodiments 147 to 153, wherein the coding sequence encoding the BAG3 protein has at least 80% sequence identity to SEQ ID NO:151 . 155. The rAAV of any one of embodiments 147 to 153, wherein the coding sequence encoding the BAG3 protein has at least 85% sequence identity to SEQ ID NO:151 .

156. The rAAV of any one of embodiments 147 to 153, wherein the coding sequence encoding the BAG3 protein has at least 90% sequence identity to SEQ ID NO:151 .

157. The rAAV of any one of embodiments 147 to 153, wherein the coding sequence encoding the BAG3 protein has at least 95% sequence identity to SEQ ID NO:151 .

158. The rAAV of any one of embodiments 147 to 153, wherein the coding sequence encoding the BAG3 protein has at least 96% sequence identity to SEQ ID NO:151 .

159. The rAAV of any one of embodiments 147 to 153, wherein the coding sequence encoding the BAG3 protein has at least 97% sequence identity to SEQ ID NO:151 .

160. The rAAV of any one of embodiments 147 to 153, wherein the coding sequence encoding the BAG3 protein has at least 98% sequence identity to SEQ ID NO:151 .

161 . The rAAV of any one of embodiments 147 to 153, wherein the coding sequence encoding the BAG3 protein has at least 99% sequence identity to SEQ ID NO:151 .

162. The rAAV of any one of embodiments 147 to 153, wherein the coding sequence encoding the BAG3 protein has 100% sequence identity to SEQ ID NO:151.

163. The rAAV of any one of embodiments 1 to 111 , wherein the polynucleotide comprises a coding sequence encoding a MYBPC3 protein.

164. The rAAV of embodiment 163, wherein the MYBPC3 protein is a human MYBPC3 protein.

165. The rAAV of embodiment 163 or embodiment 164, wherein the MYBPC3 protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NQ:200.

166. The rAAV of embodiment 163 or embodiment 164, wherein the MYBPC3 protein comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NQ:200.

167. The rAAV of embodiment 163 or embodiment 164, wherein the MYBPC3 protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NQ:200.

168. The rAAV of embodiment 163 or embodiment 164, wherein the MYBPC3 protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NQ:200.

169. The rAAV of embodiment 163 or embodiment 164, wherein the MYBPC3 protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NQ:200. 170. The rAAV of embodiment 163 or embodiment 164, wherein the MYBPC3 protein comprises an amino acid sequence having 100% sequence identity to SEQ ID N0:200.

171 . The rAAV of embodiment 163 or embodiment 164, wherein the MYBPC3 protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:201 .

172. The rAAV of embodiment 163 or embodiment 164, wherein the MYBPC3 protein comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NQ:201 .

173. The rAAV of embodiment 163 or embodiment 164, wherein the MYBPC3 protein comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NQ:201 .

174. The rAAV of embodiment 163 or embodiment 164, wherein the MYBPC3 protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NQ:201 .

175. The rAAV of embodiment 163 or embodiment 164, wherein the MYBPC3 protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NQ:201 .

176. The rAAV of embodiment 163 or embodiment 164, wherein the MYBPC3 protein comprises an amino acid sequence having 100% sequence identity to SEQ ID NQ:201.

177. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 80% sequence identity to SEQ ID NQ:202.

178. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 85% sequence identity to SEQ ID NQ:202.

179. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 90% sequence identity to SEQ ID NQ:202.

180. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 95% sequence identity to SEQ ID NQ:202.

181 . The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 96% sequence identity to SEQ ID NQ:202.

182. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 97% sequence identity to SEQ ID NQ:202.

183. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 98% sequence identity to SEQ ID NQ:202.

184. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 98% sequence identity to SEQ ID NQ:202. 185. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has 100% sequence identity to SEQ ID NO:202.

186. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 80% sequence identity to SEQ ID NO:203.

187. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 85% sequence identity to SEQ ID NQ:203.

188. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 90% sequence identity to SEQ ID NQ:203.

189. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 95% sequence identity to SEQ ID NQ:203.

190. The rAAV of any one of embodiments 163to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 96% sequence identity to SEQ ID NQ:203.

191 . The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 97% sequence identity to SEQ ID NQ:203.

192. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 98% sequence identity to SEQ ID NQ:203.

193. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has at least 98% sequence identity to SEQ ID NQ:203.

194. The rAAV of any one of embodiments 163 to 170, wherein the coding sequence encoding the MYBPC3 protein has 100% sequence identity to SEQ ID NQ:203.

195. The rAAV of any one of embodiments 1 to 194, wherein the polynucleotide further comprises one or more expression regulatory elements operably linked to the coding sequence.

196. The rAAV of embodiment 195, wherein the one or more expression regulatory elements comprise a promoter.

197. The rAAV of embodiment 196, wherein the promoter is a muscle-specific promoter.

198. The rAAV of any one of embodiments 196 to 197, wherein the promoter is a myosin light chain 2 (MLC-2) promoter.

199. The rAAV of embodiment 198, wherein the MLC-2 promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:300. 200. The rAAV of embodiment 199, wherein the MLC-2 promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:300.

201 . The rAAV of embodiment 199, wherein the MLC-2 promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID N0:300.

202. The rAAV of embodiment 199, wherein the MLC-2 promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:300.

203. The rAAV of embodiment 199, wherein the MLC-2 promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:300.

204. The rAAV of embodiment 199, wherein the MLC-2 promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:300.

205. The rAAV of embodiment 199, wherein the MLC-2 promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:300.

206. The rAAV of embodiment 199, wherein the MLC-2 promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:300.

207. The rAAV of any one of embodiments 196 to 197, wherein the promoter is a BAG3 promoter.

208. The rAAV of embodiment 207, wherein the BAG3 promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:301.

209. The rAAV of embodiment 208, wherein the BAG3 promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:301 .

210. The rAAV of embodiment 208, wherein the BAG3 promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:301 .

211 . The rAAV of embodiment 208, wherein the BAG3 promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:301 .

212. The rAAV of embodiment 208, wherein the BAG3 promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:301 .

213. The rAAV of embodiment 208, wherein the BAG3 promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:301 .

214. The rAAV of embodiment 208, wherein the BAG3 promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:301 . 215. The rAAV of embodiment 208, wherein the BAG3 promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO:301.

216. The rAAV of any one of embodiments 196 to 197, wherein the promoter is a CK8e promoter.

217. The rAAV of embodiment 216, wherein the CK8e promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:302.

218. The rAAV of embodiment 217, wherein the CK8e promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:302.

219. The rAAV of embodiment 217, wherein the CK8e promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:302.

220. The rAAV of embodiment 217, wherein the CK8e promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:302.

221 . The rAAV of embodiment 217, wherein the CK8e promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:302.

222. The rAAV of embodiment 217, wherein the CK8e promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:302.

223. The rAAV of embodiment 217, wherein the CK8e promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:302.

224. The rAAV of embodiment 217, wherein the CK8e promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:302.

225. The rAAV of any one of embodiments 196 to 197, wherein the promoter is a MSEC-725a promoter.

226. The rAAV of embodiment 225, wherein the MSEC-725a promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:303.

227. The rAAV of embodiment 226, wherein the MSEC-725a promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:303.

228. The rAAV of embodiment 226, wherein the MSEC-725a promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:303. 229. The rAAV of embodiment 226, wherein the MSEC-725a promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NO:303.

230. The rAAV of embodiment 226, wherein the MSEC-725a promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NO:303.

231 . The rAAV of embodiment 226, wherein the MSEC-725a promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:303.

232. The rAAV of embodiment 226, wherein the MSEC-725a promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:303.

233. The rAAV of embodiment 226, wherein the MSEC-725a promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:303.

234. The rAAV of any one of embodiments 196 to 197, wherein the promoter is a CK7 promoter.

235. The rAAV of embodiment 234, wherein the CK7 promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:304.

236. The rAAV of embodiment 235, wherein the CK7 promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:304.

237. The rAAV of embodiment 235, wherein the CK7 promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:304.

238. The rAAV of embodiment 235, wherein the CK7 promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:304.

239. The rAAV of embodiment 235, wherein the CK7 promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:304.

240. The rAAV of embodiment 235, wherein the CK7 promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:304.

241 . The rAAV of embodiment 235, wherein the CK7 promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:304.

242. The rAAV of embodiment 235, wherein the CK7 promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:304.

243. The rAAV of any one of embodiments 196 to 197, wherein the promoter is a MHCK7 promoter. 244. The rAAV of embodiment 243, wherein the MHCK7 promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:305.

245. The rAAV of embodiment 244, wherein the MHCK7 promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:305.

246. The rAAV of embodiment 244, wherein the MHCK7 promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:305.

247. The rAAV of embodiment 244, wherein the MHCK7 promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:305.

248. The rAAV of embodiment 244, wherein the MHCK7 promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:305.

249. The rAAV of embodiment 244, wherein the MHCK7 promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:305.

250. The rAAV of embodiment 244, wherein the MHCK7 promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:305.

251 . The rAAV of embodiment 244, wherein the MHCK7 promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:305.

252. The rAAV of any one of embodiments 196 to 197, wherein the promoter is a MYH7 promoter.

253. The rAAV of embodiment 252, wherein the MYH7 promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:306.

254. The rAAV of embodiment 253, wherein the MYH7promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:306.

255. The rAAV of embodiment 253, wherein the MYH7 promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:306.

256. The rAAV of embodiment 253, wherein the MYH7 promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:306.

257. The rAAV of embodiment 253, wherein the MYH7 promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:306. 258. The rAAV of embodiment 253, wherein the MYH7 promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NO:306.

259. The rAAV of embodiment 253, wherein the MYH7 promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NO:306.

260. The rAAV of embodiment 253, wherein the MYH7 promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:306.

261 . The rAAV of any one of embodiments 196 to 197, wherein the promoter is a CSRP3 promoter.

262. The rAAV of embodiment 261 , wherein the CSRP3 promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:307.

263. The rAAV of embodiment 262, wherein the CSRP3 promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:307.

264. The rAAV of embodiment 262, wherein the CSRP3 promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:307.

265. The rAAV of embodiment 262, wherein the CSRP3 promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:307.

266. The rAAV of embodiment 262, wherein the CSRP3 promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:307.

267. The rAAV of embodiment 262, wherein the CSRP3 promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:307.

268. The rAAV of embodiment 262, wherein the CSRP3 promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:307.

269. The rAAV of embodiment 262, wherein the CSRP3 promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:307.

270. The rAAV of any one of embodiments 196 to 197, wherein the promoter is a HRC promoter.

271 . The rAAV of embodiment 270, wherein the HRC promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:308. 272. The rAAV of embodiment 271 , wherein the HRC promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:308.

273. The rAAV of embodiment 271 , wherein the HRC promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:308.

274. The rAAV of embodiment 271 , wherein the HRC promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:308.

275. The rAAV of embodiment 271 , wherein the HRC promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:308.

276. The rAAV of embodiment 271 , wherein the HRC promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:308.

277. The rAAV of embodiment 271 , wherein the HRC promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:308.

278. The rAAV of embodiment 271 , wherein the HRC promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:308.

279. The rAAV of any one of embodiments 196 to 197, wherein the promoter is a MYOZ2 promoter.

280. The rAAV of embodiment 279, wherein the MYOZ2 promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NQ:309.

281 . The rAAV of embodiment 280, wherein the MYOZ2 promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:309.

282. The rAAV of embodiment 280, wherein the MYOZ2 promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:309.

283. The rAAV of embodiment 280, wherein the MYOZ2 promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:309.

284. The rAAV of embodiment 280, wherein the MYOZ2 promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:309.

285. The rAAV of embodiment 280, wherein the MYOZ2 promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:309.

286. The rAAV of embodiment 280, wherein the MYOZ2 promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:309. 287. The rAAV of embodiment 280, wherein the MYOZ2 promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO:309.

288. The rAAV of any one of embodiments 196 to 197, wherein the promoter is a TNNT2 promoter.

289. The rAAV of embodiment 288, wherein the TNNT2 promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:310.

290. The rAAV of embodiment 289, wherein the TNNT2 promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NQ:310.

291 . The rAAV of embodiment 289, wherein the TNNT2 promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:310.

292. The rAAV of embodiment 289, wherein the TNNT2 promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:310.

293. The rAAV of embodiment 289, wherein the TNNT2 promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:310.

294. The rAAV of embodiment 289, wherein the TNNT2 promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:310.

295. The rAAV of embodiment 289, wherein the TNNT2 promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:310.

296. The rAAV of embodiment 289, wherein the TNNT2 promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:310.

297. The rAAV of embodiment 288, wherein the TNNT2 promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:311 .

298. The rAAV of embodiment 297, wherein the TNNT2 promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:311 .

299. The rAAV of embodiment 297, wherein the TNNT2 promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:311 .

300. The rAAV of embodiment 297, wherein the TNNT2 promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NO:311 . 301 . The rAAV of embodiment 297, wherein the TNNT2 promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NO:311 .

302. The rAAV of embodiment 297, wherein the TNNT2 promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NO:311 .

303. The rAAV of embodiment 297, wherein the TNNT2 promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NO:311 .

304. The rAAV of embodiment 297, wherein the TNNT2 promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO:311.

305. The rAAV of any one of embodiments 196 to 197, wherein the promoter is a aMHC promoter.

306. The rAAV of embodiment 305, wherein the aMHC promoter comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:312.

307. The rAAV of embodiment 306, wherein the aMHC promoter comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:312.

308. The rAAV of embodiment 306, wherein the aMHC promoter comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:312.

309. The rAAV of embodiment 306, wherein the aMHC promoter comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NO:312.

310. The rAAV of embodiment 306, wherein the aMHC promoter comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NO:312.

311 . The rAAV of embodiment 306, wherein the aMHC promoter comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NO:312.

312. The rAAV of embodiment 306, wherein the aMHC promoter comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NO:312.

313. The rAAV of embodiment 306, wherein the aMHC promoter comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO:312.

314. The rAAV of any one of embodiments 195 to 313, wherein the one or more expression regulatory elements comprises a posttranscriptional regulatory element sequence. 315. The rAAV of embodiment 314, wherein the posttranscriptional regulatory element sequence comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID N0:400.

316. The rAAV of embodiment 315, wherein the posttranscriptional regulatory element sequence comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID N0:400.

317. The rAAV of embodiment 315, wherein the posttranscriptional regulatory element sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NQ:400.

318. The rAAV of embodiment 315, wherein the posttranscriptional regulatory element sequence comprises a nucleotide sequence having at least 96% sequence identity to SEQ ID NQ:400.

319. The rAAV of embodiment 315, wherein the posttranscriptional regulatory element sequence comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NQ:400.

320. The rAAV of embodiment 315, wherein the posttranscriptional regulatory element sequence comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NQ:400.

321 . The rAAV of embodiment 315, wherein the posttranscriptional regulatory element sequence comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NQ:400.

322. The rAAV of embodiment 315, wherein the posttranscriptional regulatory element sequence comprises a nucleotide sequence having 100% sequence identity to SEQ ID NQ:400.

323. The rAAV of any one of embodiments 195 to 322, wherein the polynucleotide further comprises a BAG3 5’ UTR’ sequence.

324. The rAAV of embodiment 323, wherein the BAG3 5’ UTR sequence comprises the nucleotide sequence of SEQ ID NQ:500.

325. The rAAV of embodiment 323, wherein the BAG3 5’ UTR sequence comprises the nucleotide sequence of SEQ ID NQ:501 .

326. The rAAV of any one of embodiments 195 to 325, wherein the polynucleotide further comprises a BAG3 3’ UTR’ sequence.

327. The rAAV of embodiment 326, wherein the BAG3 3’ UTR sequence comprises the nucleotide sequence of SEQ ID NQ:502.

328. The rAAV of any one of embodiments 195 to 327, wherein the polynucleotide further comprises a polyadenylation signal sequence.

329. The rAAV of embodiment 328, wherein the polyadenylation signal sequence comprises a bovine growth hormone (BGH) polyadenylation signal sequence. 330. The rAAV of embodiment 329, wherein the nucleotide sequence of the BGH polyadenylation signal sequence comprises the nucleotide sequence of SEQ ID NO:401.

331 . The rAAV of embodiment 328, wherein the polyadenylation signal sequence comprises the nucleotide sequence of SEQ ID NO:402.

332. The rAAV of any one of embodiments 1 to 331 , wherein the polynucleotide further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, optionally wherein the 5’ ITR comprises a nucleotide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NQ:600, SEQ ID NQ:601 , SEQ ID NQ:602, or SEQ ID NQ:603, and/or wherein the 3’ ITR comprises a nucleotide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NQ:604 or SEQ ID NQ:605.

333. The rAAV of embodiment 332, wherein the nucleotide sequence of the 5’ ITR comprises the nucleotide sequence of SEQ ID NQ:600.

334. The rAAV of embodiment 332, wherein the nucleotide sequence of the 5’ ITR comprises the nucleotide sequence of SEQ ID NQ:601.

335. The rAAV of embodiment 332, wherein the nucleotide sequence of the 5’ ITR comprises the nucleotide sequence of SEQ ID NQ:602.

336. The rAAV of embodiment 332, wherein the nucleotide sequence of the 5’ ITR comprises the nucleotide sequence of SEQ ID NQ:603.

337. The rAAV of any one of embodiments 332 to 336, wherein the nucleotide sequence of the 3’ ITR comprises the nucleotide sequence of SEQ ID NQ:604.

338. The rAAV of any one of embodiments 332 to 336, wherein the nucleotide sequence of the 3’ ITR comprises the nucleotide sequence of SEQ ID NQ:605.

339. The rAAV of embodiment 332, wherein the 5’ ITR comprises a nucleotide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NQ:600, SEQ ID NQ:601 , SEQ ID NQ:602, or SEQ ID NQ:603, and/or wherein the 3’ ITR comprises a nucleotide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NQ:604, SEQ ID NQ:605, SEQ ID NQ:606, SEQ ID NQ:607, SEQ ID NQ:608, SEQ ID NQ:609, or SEQ ID NQ:610.

340. The rAAV of embodiment 339, wherein the nucleotide sequence of the 5’ ITR comprises the nucleotide sequence of SEQ ID NQ:600.

341 . The rAAV of embodiment 339, wherein the nucleotide sequence of the 5’ ITR comprises the nucleotide sequence of SEQ ID NQ:601. 342. The rAAV of embodiment 339, wherein the nucleotide sequence of the 5’ ITR comprises the nucleotide sequence of SEQ ID NO:602.

343. The rAAV of embodiment 339, wherein the nucleotide sequence of the 5’ ITR comprises the nucleotide sequence of SEQ ID NO:603.

344. The rAAV of any one of embodiments 339 to 343, wherein the nucleotide sequence of the 3’ ITR comprises the nucleotide sequence of SEQ ID NQ:604.

345. The rAAV of any one of embodiments 339 to 343, wherein the nucleotide sequence of the 3’ ITR comprises the nucleotide sequence of SEQ ID NQ:605.

346. The rAAV of any one of embodiments 339 to 343, wherein the nucleotide sequence of the 3’ ITR comprises the nucleotide sequence of SEQ ID NQ:606.

347. The rAAV of any one of embodiments 339 to 343, wherein the nucleotide sequence of the 3’ ITR comprises the nucleotide sequence of SEQ ID NQ:607.

348. The rAAV of any one of embodiments 339 to 343, wherein the nucleotide sequence of the 3’ ITR comprises the nucleotide sequence of SEQ ID NQ:608.

349. The rAAV of any one of embodiments 339 to 343, wherein the nucleotide sequence of the 3’ ITR comprises the nucleotide sequence of SEQ ID NQ:609.

350. The rAAV of any one of embodiments 339 to 343, wherein the nucleotide sequence of the 3’ ITR comprises the nucleotide sequence of SEQ ID NO:610.

351 . The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:800.

352. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:801.

353. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:802.

354. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:803.

355. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:804.

356. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:805. 357. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:806.

358. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:807.

359. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:808.

360. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:809.

361 . The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:810.

362. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:900.

363. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:901.

364. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:902.

365. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:903.

366. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:904.

367. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:905.

368. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:906.

369. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:907.

370. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:908.

371 . The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NQ:909. 372. The rAAV of any one of embodiments 1 to 109, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:910.

373. In a recombinant adeno-associated virus (rAAV) comprising (a) a capsid comprising a capsid protein and (b) an encapsulated polynucleotide comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein or a myosin-binding protein C, cardiactype (MYBPC3) protein, the improvement comprising including in the capsid a modified capsid protein having a targeting peptide within variable region VIII (VR VIII), wherein the targeting peptide comprises the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

374. In a recombinant adeno-associated virus (rAAV) comprising (a) a capsid comprising a capsid protein and (b) an encapsulated polynucleotide comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein or a myosin-binding protein C, cardiactype (MYBPC3) protein, the improvement comprising (i) including in the capsid a modified capsid protein having a targeting peptide within variable region VIII (VR VIII), wherein the targeting peptide comprises the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 and/or (ii) an encapsulated polynucleotide described in any one of embodiments 1 to 372.

375. The rAAV of embodiment 373 or embodiment 374, comprising a modified capsid protein and/or polynucleotide described in any one of embodiments 1 to 372.

376. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:8, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:100, optionally wherein the polynucleotide comprises (i) a BAG3 coding sequence as described in any one of embodiments 120 to 146 and 154 to 162, and/or (ii) a promoter as described in any one of embodiments 196 to 313, and/or (iii) a posttranscriptional regulatory element sequence as described in any one of embodiments 314 to 322 and/or (iv) a polyadenylation sequence as described in any one of embodiments 328 to 331 , and/or (v) a 5’ ITR as described in any one of embodiments 332 to 336 and 339 to 343, and/or (vi) a 3’ ITR as described in any one of embodiments 337 to 339 and 344 to 350.

377. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:9, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID N0:100, optionally wherein the polynucleotide comprises (i) a BAG3 coding sequence as described in any one of embodiments 120 to 146 and 154 to 162, and/or (ii) a promoter as described in any one of embodiments 196 to 313, and/or (iii) a posttranscriptional regulatory element sequence as described in any one of embodiments 314 to 322 and/or (iv) a polyadenylation sequence as described in any one of embodiments 328 to 331 , and/or (v) a 5’ ITR as described in any one of embodiments 332 to 336 and 339 to 343, and/or (vi) a 3’ ITR as described in any one of embodiments 337 to 339 and 344 to 350.

378. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:10, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID N0:100, optionally wherein the polynucleotide comprises (i) a BAG3 coding sequence as described in any one of embodiments 120 to 146 and 154 to 162, and/or (ii) a promoter as described in any one of embodiments 196 to 313, and/or (iii) a posttranscriptional regulatory element sequence as described in any one of embodiments 314 to 322 and/or (iv) a polyadenylation sequence as described in any one of embodiments 328 to 331 , and/or (v) a 5’ ITR as described in any one of embodiments 332 to 336 and 339 to 343, and/or (vi) a 3’ ITR as described in any one of embodiments 337 to 339 and 344 to 350.

379. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:11 , and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:100, optionally wherein the polynucleotide comprises (i) a BAG3 coding sequence as described in any one of embodiments 120 to 146 and 154 to 162, and/or (ii) a promoter as described in any one of embodiments 196 to 313, and/or (iii) a posttranscriptional regulatory element sequence as described in any one of embodiments 314 to 322 and/or (iv) a polyadenylation sequence as described in any one of embodiments 328 to 331 , and/or (v) a 5’ ITR as described in any one of embodiments 332 to 336 and 339 to 343, and/or (vi) a 3’ ITR as described in any one of embodiments 337 to 339 and 344 to 350.

380. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid; and b. a polynucleotide encapsulated by the capsid and comprising a promoter as described in any one of embodiments 196 to 313 operably linked a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID N0:100.

381 . The rAAV of embodiment 380, wherein the promoter is a CK8e promoter as described in any one of embodiments 216 to 224.

382. The rAAV of any one of embodiments 376 to 381 , wherein the coding sequence encodes a BAG3 protein comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID N0:100.

383. The rAAV of any one of embodiments 376 to 381 , wherein the coding sequence encodes a BAG3 protein comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NQ:100.

384. The rAAV of any one of embodiments 376 to 381 , wherein the coding sequence encodes a BAG3 protein comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NQ:100.

385. The rAAV of any one of embodiments 376 to 381 , wherein the coding sequence encodes a BAG3 protein comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NQ:100.

386. The rAAV of any one of embodiments 376 to 381 , wherein the coding sequence encodes a BAG3 protein comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NQ:100.

387. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:8, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a myosin-binding protein C, cardiac-type (MYBPC3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:200, optionally wherein the polynucleotide comprises (i) a MYBPC3 coding sequence as described in any one of embodiments 177 to 194, and/or (ii) a promoter as described in any one of embodiments 196 to 313, and/or (iii) a posttranscriptional regulatory element sequence as described in any one of embodiments 314 to 322 and/or (iv) a polyadenylation sequence as described in any one of embodiments 328 to 331 , and/or (v) a 5’ ITR as described in any one of embodiments 332 to 336 and 339 to 343, and/or (vi) a 3’ ITR as described in any one of embodiments 337 to 339 and 344 to 350.

388. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:9, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a myosin-binding protein C, cardiac-type (MYBPC3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID N0:200, optionally wherein the polynucleotide comprises (i) a MYBPC3 coding sequence as described in any one of embodiments 177 to 194, and/or (ii) a promoter as described in any one of embodiments 196 to 313, and/or (iii) a posttranscriptional regulatory element sequence as described in any one of embodiments 314 to 322 and/or (iv) a polyadenylation sequence as described in any one of embodiments 328 to 331 , and/or (v) a 5’ ITR as described in any one of embodiments 332 to 336 and 339 to 343, and/or (vi) a 3’ ITR as described in any one of embodiments 337 to 339 and 344 to 350.

389. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:10, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a myosin-binding protein C, cardiac-type (MYBPC3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:200, optionally wherein the polynucleotide comprises (i) a MYBPC3 coding sequence as described in any one of embodiments 177 to 194, and/or (ii) a promoter as described in any one of embodiments 196 to 313, and/or (iii) a posttranscriptional regulatory element sequence as described in any one of embodiments 314 to 322 and/or (iv) a polyadenylation sequence as described in any one of embodiments 328 to 331 , and/or (v) a 5’ ITR as described in any one of embodiments 332 to 336 and 339 to 343, and/or (vi) a 3’ ITR as described in any one of embodiments 337 to 339 and 344 to 350.

390. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:11 , and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a myosin-binding protein C, cardiac-type (MYBPC3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:200, optionally wherein the polynucleotide comprises (i) a MYBPC3 coding sequence as described in any one of embodiments 177 to 194, and/or (ii) a promoter as described in any one of embodiments 196 to 313, and/or (iii) a posttranscriptional regulatory element sequence as described in any one of embodiments 314 to 322 and/or (iv) a polyadenylation sequence as described in any one of embodiments 328 to 331 , and/or (v) a 5’ ITR as described in any one of embodiments 332 to 336 and 339 to 343, and/or (vi) a 3’ ITR as described in any one of embodiments 337 to 339 and 344 to 350. 391 . A recombinant adeno-associated virus (rAAV) comprising: a. a capsid; and b. a polynucleotide encapsulated by the capsid and comprising a promoter as described in any one of embodiments 196 to 313 operably linked a coding sequence encoding a myosin- binding protein C, cardiac-type (MYBPC3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID N0:200.

392. The rAAV of embodiment 391 , wherein the promoter is a CK8e promoter as described in any one of embodiments 216 to 224.

393. The rAAV of any one of embodiments 387 to 392, wherein the coding sequence encodes a MYBCP3 protein comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID N0:200.

394. The rAAV of any one of embodiments 387 to 392, wherein the coding sequence encodes a MYBCP3 protein comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NQ:200.

395. The rAAV of any one of embodiments 387 to 392, wherein the coding sequence encodes a MYBCP3 protein comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NQ:200.

396. The rAAV of any one of embodiments 387 to 392, wherein the coding sequence encodes a MYBCP3 protein comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NQ:200.

397. The rAAV of any one of embodiments 387 to 392, wherein the coding sequence encodes a MYBCP3 protein comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NQ:200.

398. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:8, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a myosin-binding protein C, cardiac-type (MYBPC3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:201 , optionally wherein the polynucleotide comprises (i) a promoter as described in any one of embodiments 196 to 313, and/or (ii) a posttranscriptional regulatory element sequence as described in any one of embodiments 314 to 322 and/or (iii) a polyadenylation sequence as described in any one of embodiments 328 to 331 , and/or (iv) a 5’ ITR as described in any one of embodiments 332 to 336 and 339 to 343, and/or (vi) a 3’ ITR as described in any one of embodiments 337 to 339 and 344 to 350. 399. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:9, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a myosin-binding protein C, cardiac-type (MYBPC3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:201 , optionally wherein the polynucleotide comprises (i) a promoter as described in any one of embodiments 196 to 313, and/or (ii) a posttranscriptional regulatory element sequence as described in any one of embodiments 314 to 322 and/or (iii) a polyadenylation sequence as described in any one of embodiments 328 to 331 , and/or (iv) a 5’ ITR as described in any one of embodiments 332 to 336 and 339 to 343, and/or (vi) a 3’ ITR as described in any one of embodiments 337 to 339 and 344 to 350.

400. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:10, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a myosin-binding protein C, cardiac-type (MYBPC3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:201 , optionally wherein the polynucleotide comprises (i) a promoter as described in any one of embodiments 196 to 313, and/or (ii) a posttranscriptional regulatory element sequence as described in any one of embodiments 314 to 322 and/or (iii) a polyadenylation sequence as described in any one of embodiments 328 to 331 , and/or (iv) a 5’ ITR as described in any one of embodiments 332 to 336 and 339 to 343, and/or (vi) a 3’ ITR as described in any one of embodiments 337 to 339 and 344 to 350.

401 . A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:11 , and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a myosin-binding protein C, cardiac-type (MYBPC3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NQ:201 , optionally wherein the polynucleotide comprises (i) a promoter as described in any one of embodiments 196 to 313, and/or (ii) a posttranscriptional regulatory element sequence as described in any one of embodiments 314 to 322 and/or (iii) a polyadenylation sequence as described in any one of embodiments 328 to 331 , and/or (iv) a 5’ ITR as described in any one of embodiments 332 to 336 and 339 to 343, and/or (vi) a 3’ ITR as described in any one of embodiments 337 to 339 and 344 to 350. 402. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid; and b. a polynucleotide encapsulated by the capsid and comprising a promoter as described in any one of embodiments 196 to 313 operably linked a coding sequence encoding a myosin- binding protein C, cardiac-type (MYBPC3) protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:201 .

403. The rAAV of embodiment 402, wherein the promoter is a CK8e promoter as described in any one of embodiments 216 to 224.

404. The rAAV of any one of embodiments 398 to 403, wherein the coding sequence encodes a MYBCP3 protein comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:201.

405. The rAAV of any one of embodiments 398 to 403, wherein the coding sequence encodes a MYBCP3 protein comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NQ:201.

406. The rAAV of any one of embodiments 398 to 403, wherein the coding sequence encodes a MYBCP3 protein comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NQ:201.

407. The rAAV of any one of embodiments 398 to 403, wherein the coding sequence encodes a MYBCP3 protein comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NQ:201.

408. The rAAV of any one of embodiments 398 to 403, wherein the coding sequence encodes a MYBCP3 protein comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NQ:201.

409. The rAAV of any one of embodiments 1 to 408, which comprises a self-complementary rAAV genome.

410. A polynucleotide comprising a nucleotide sequence as set forth in any one of SEQ ID NQS:800-810 and 900-910.

411. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:801 .

412. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:802.

413. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:803.

414. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:804. 415. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO:805.

416. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO:806.

417. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:807.

418. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:808.

419. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:809.

420. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:810.

421 . A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:900.

422. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:901 .

423. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:902.

424. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:903.

425. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:904.

426. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:905.

427. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:906.

428. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:907.

429. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:908.

430. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:909.

431 . A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:910.

432. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid; and b. a polynucleotide according to any one of embodiments 410 to 431 encapsulated by the capsid.

433. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:8, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a nucleotide sequence as set forth in SEQ ID NQ:810. 434. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:9, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a nucleotide sequence as set forth in SEQ ID NO:810.

435. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:10, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a nucleotide sequence as set forth in SEQ ID NQ:810.

436. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:1 1 , and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a nucleotide sequence as set forth in SEQ ID NQ:810.

437. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:8, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a nucleotide sequence as set forth in SEQ ID NQ:910.

438. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:9, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a nucleotide sequence as set forth in SEQ ID NQ:910.

439. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:10, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a nucleotide sequence as set forth in SEQ ID NQ:910.

440. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:11 , and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a nucleotide sequence as set forth in SEQ ID NO:910.

441 . A pharmaceutical composition comprising the rAAV of any one of embodiments 1 to 409 and 432 to 440 and a pharmaceutically acceptable excipient.

442. A unit dose comprising the pharmaceutical composition of embodiment 441 .

443. A host cell comprising the polynucleotide of any one of embodiments 410 to 431 .

444. A host cell engineered to produce the rAAV of any one of embodiments 1 to 409 and 432 to 440.

445. The host cell of embodiment 444, which comprises a polynucleotide expressing one or more capsid proteins of the rAAV, a functional rep gene, and a recombinant nucleic acid vector comprising AAV ITRs and the coding sequence operably linked to a promoter.

446. A method of transferring a polynucleotide to the heart of a subject comprising administering the rAAV of any one of embodiments 1 to 409 and 432 to 440, the pharmaceutical composition of embodiment 441 or the unit dose of embodiment 442 to the subject.

447. The method of embodiment 446, wherein the subject has or is at risk of a cardiac disease.

448. A method of treating a subject having or at risk of a cardiac disease comprising administering a therapeutically effective amount of the rAAV of any one of embodiments 1 to 409 and 432 to 440, the pharmaceutical composition of embodiment 441 or the unit dose of embodiment 442 to the subject.

449. The method of embodiment 447 or embodiment 448, wherein the subject has a cardiac disease (e.g., due to a BAG3 or MYBPC3 mutation).

450. The method of embodiment 447 or embodiment 448, wherein the subject is at risk of a cardiac disease (e.g., due to a BAG3 or MYBPC3 mutation).

451 . The method of any one of embodiments 447 to 450, wherein the cardiac disease is a genetic cardiomyopathy, dilated cardiomyopathy (DCM) (e.g., idiopathic DCM, familial DCM, or non- familial DCM), hypertrophic cardiomyopathy, non-ischemic cardiomyopathy, heart failure (e.g., congestive heart failure, heart failure due to reduced ejection fraction, heart failure due to coronary artery disease, acute heart failure, or end-stage heart failure), restrictive cardiomyopathy, left-ventricular noncompaction, atherosclerosis, coronary artery disease, ischemic heart disease, myocarditis, hypertensive heart disease, valvular disease, congenital heart disease, or myocardial infarction. 452. The method of any one of embodiments 447 to 450, wherein the cardiac disease is a genetic cardiomyopathy.

453. The method of any one of embodiments 447 to 450, wherein the cardiac disease is dilated cardiomyopathy (DCM).

454. The method of embodiment 453, wherein the DCM is idiopathic DCM.

455. The method of embodiment 453, wherein the DCM is familial DCM.

456. The method of embodiment 453, wherein the DCM is non-familial DCM.

457. The method of any one of embodiments 447 to 450, wherein the cardiac disease is nonischemic cardiomyopathy.

458. The method of any one of embodiments 447 to 450, wherein the cardiac disease is heart failure.

459. The method of any one of embodiments 447 to 450, wherein the cardiac disease is heart failure due to reduced ejection fraction.

460. The method of any one of embodiments 447 to 450, wherein the cardiac disease is heart failure due to coronary artery disease.

461 . The method of any one of embodiments 447 to 450, wherein the cardiac disease is hypertrophic cardiomyopathy.

462. The method of any one of embodiments 447 to 450, wherein the cardiac disease is restrictive cardiomyopathy.

463. The method of any one of embodiments 447 to 450, wherein the cardiac disease is left- ventricular non-compaction.

464. The method of any one of embodiments 447 to 450, wherein the cardiac disease is atherosclerosis.

465. The method of any one of embodiments 447 to 450, wherein the cardiac disease is coronary artery disease.

466. The method of any one of embodiments 447 to 450, wherein the cardiac disease is ischemic heart disease.

467. The method of any one of embodiments 447 to 450, wherein the cardiac disease is myocarditis. 468. The method of any one of embodiments 447 to 450, wherein the cardiac disease is hypertensive heart disease.

469. The method of any one of embodiments 447 to 450, wherein the cardiac disease is valvular disease.

470. The method of any one of embodiments 447 to 450, wherein the cardiac disease is congenital heart disease.

471 . The method of any one of embodiments 447 to 450, wherein the cardiac disease is myocardial infarction.

472. The method of any one of embodiments 447 to 450, wherein the cardiac disease is congestive heart failure.

473. The method of any one of embodiments 446 to 472, wherein the subject has a mutated BAG3 gene.

474. The method of any one of embodiments 446 to 472, wherein the subject has a mutated MYBPC3 gene.

475. The method of any one of embodiments 446 to 474, wherein the rAAV, pharmaceutical composition or unit dose is administered systemically.

476. The method of embodiment 475, wherein the rAAV, pharmaceutical composition or unit dose is administered intravenously.

477. The method of any one of embodiments 446 to 476, which comprises administering a 6e11 vg/kg to 1e14 vg/kg dose of rAAV to the subject, e.g., 6e11 vg/kg, 1e12 vg/kg, 2e12 vg/kg, 1 e13 vg/kg, 1.5e13 vg/kg, or 1e14 vg/kg.

478. The method of any one of embodiments 446 to 476, which comprises administering a 1 e12 vg/kg to 1 e14 vg/kg dose of rAAV to the subject.

479. The method of any one of embodiments 446 to 476, which comprises administering a 1 ,5e12 vg/kg to 1 ,5e13 vg/kg dose of rAAV to the subject, e.g., 1e13 vg/kg.

480. The method of any one of embodiments 446 to 476, which comprises administering a 1e13 vg/kg to 1e14 vg/kg dose of rAAV to the subject, e.g., 3e13 vg/kg.

481 . The rAAV of any one of embodiments 1 to 409 and 432 to 440, the pharmaceutical composition of embodiment 441 , or the unit dose of embodiment 442 for use in a method of transferring a polynucleotide to the heart of a subject. 482. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to embodiment 481 , wherein the subject has or is at risk of a cardiac disease.

483. The rAAV of any one of embodiments 1 to 409 and 432 to 440, the pharmaceutical composition of embodiment 441 , or the unit dose of embodiment 442 for use in a method of treating a subject having or at risk of a cardiac disease.

484. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to embodiment 482 or embodiment 483, wherein the subject has a cardiac disease (e.g., due to a BAG3 or MYBPC3 mutation).

485. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to embodiment 482 or embodiment 483, wherein the subject is at risk of a cardiac disease (e.g., due to a BAG3 or MYBPC3 mutation).

486. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is a genetic cardiomyopathy, dilated cardiomyopathy (DCM) (e.g., idiopathic DCM, familial DCM, or non-familial DCM), hypertrophic cardiomyopathy, non-ischemic cardiomyopathy, heart failure (e.g., congestive heart failure, heart failure due to reduced ejection fraction, heart failure due to coronary artery disease, acute heart failure, or endstage heart failure), restrictive cardiomyopathy, left-ventricular non-compaction, atherosclerosis, coronary artery disease, ischemic heart disease, myocarditis, hypertensive heart disease, valvular disease, congenital heart disease, or myocardial infarction.

487. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is a genetic cardiomyopathy.

488. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is dilated cardiomyopathy (DCM).

489. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to embodiment 488, wherein the DCM is idiopathic DCM.

490. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to embodiment 488, wherein the DCM is familial DCM.

491 . The rAAV for use, pharmaceutical composition for use, or unit dose for use according to embodiment 488, wherein the DCM is non-familial DCM.

492. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is non-ischemic cardiomyopathy.

493. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is heart failure. 494. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is heart failure due to reduced ejection fraction.

495. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is heart failure due to coronary artery disease.

496. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is hypertrophic cardiomyopathy.

497. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is restrictive cardiomyopathy.

498. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is left-ventricular non-compaction.

499. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is atherosclerosis.

500. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is coronary artery disease.

501 . The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is ischemic heart disease.

502. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is myocarditis.

503. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is hypertensive heart disease.

504. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is valvular disease.

505. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is congenital heart disease.

506. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is myocardial infarction.

507. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 482 to 485, wherein the cardiac disease is congestive heart failure. 508. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 481 to 507, wherein the subject has a mutated BAG3 gene.

509. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 481 to 507, wherein the subject has a mutated MYBPC3 gene.

510. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 481 to 509, wherein the method comprises administering the rAAV, pharmaceutical composition or unit dose systemically.

511 . The rAAV for use, pharmaceutical composition for use, or unit dose for use according to embodiment 510, wherein the method comprises administering the rAAV, pharmaceutical composition or unit dose intravenously.

512. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 481 to 511 , wherein the method comprises administering a 6e11 vg/kg to 1e14 vg/kg dose of rAAV to the subject, e.g., 6e11 vg/kg, 1e12 vg/kg, 2e12 vg/kg, 1e13 vg/kg, 1.5e13 vg/kg, or 1e14 vg/kg.

513. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 481 to 511 , wherein the method comprises administering a 1e12 vg/kg to 1e14 vg/kg dose of rAAV to the subject.

514. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 481 to 511 , wherein the method comprises administering a 1.5e12 vg/kg to

1 ,5e13 vg/kg dose of rAAV to the subject, e.g., 1e13 vg/kg.

515. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of embodiments 481 to 511 , wherein the method comprises administering a 1e13 vg/kg to 1e14 vg/kg dose of rAAV to the subject, e.g., 3e13 vg/kg.

8. EXAMPLES

8.1. Materials and Methods

8.1.1. Murine cKO and KI models of cardiomyopathy

8.1.1.1 BAG3 cKO model

[0184] A mouse model of BAG3 haplo-insufficiency that mirrors the observations in patients with heart failure due to BAG3 haplo-insufficiency was generated. This model utilizes a cardiac-specific heterozygous (+/-) deletion in BAG3. Mice with one allele of BAG3 flanked by loxP recombination sites (BAG3^fl/+) were crossed with a-MHC-Cre mice to generate conditional KO mice with cardiac-specific haplo-insufficiency (BAG3 cKO^+/). This mouse model with cardiomyocyte-restricted heterozygous BAG3 expression has been previously characterized and has been reported to show changes in cardiac morphology, function, and gene expression relative to wild type control mice and reflect the impact of BAG3 haplo-insufficiency on dilated cardiomyopathy (Myers et al., 2018 J Cell Phsiol. 233(9):6319-26; Fang et al., 2017, J Clin Invest. 127(8):3189-200; Myers et al., 2018 JAMA Cardiol. 3(10):929-38; Wang et al., 2023, JACC Basic Transl Sci. 8(7):820-39).

8.1.1.2 Murine MYBPC3 KI model

[0185] A MYBPC3 KI mouse model described in Vignier et al., 2009, Circ Res. 105(3):239-48 was used. These mice harbor a single point mutation (G > A transition) located on the last nucleotide of exon 6, a mutation associated with a severe phenotype in humans (Richard et al., 2003, Circulation. 107(17):2227- 32). The molecular consequence of this mutation is the production of 3 different mutant mRNAs and proteins; with total Mybpc3 mRNA and cMyBP-C protein levels markedly lower than the respective WT levels mice (Vignier et al., 2009, Circ Res. 105(3):239-48).

[0186] In the homozygous state MYBPC3 KI mouse model manifests many cardiovascular features of human HCM, including left ventricular hypertrophy, reduced fractional shortening, interstitial fibrosis and significantly reduced cMyBP-C protein levels (approximately 80% less than WT) in the heart (Vignier et al., 2009, Circ Res. 105(3):239-48). Left ventricular hypertrophy and decrease in LV contractile function appears to remain stable over time and despite the cardiac dysfunction the MYBPC3 KI mouse model has a normal life expectancy. Heterozygous MYBPC3 +/- mice have no cardiovascular phenotype.

8.1.2. Murine myocardial infarction (Ml) model

[0187] Myocardial infarction (Ml) was surgically induced in mice by a distal ligation of the left anterior descending (LAD) coronary artery creating sustained ischemia and infarction in the LV anterior wall. Cardiac infarction was confirmed by weekly echocardiography. Six weeks following surgery, when Ml mice showed significantly diminished LV function compared to WT control mice (no surgery), Ml mice received IV injection of rAAV. Global LV function was evaluated in all mice after light sedation (2% isoflurane) using a Vevo™ 770 imaging system and a scan head (VisualSonics, Miami, Florida). The left ventricular ejection fraction (LVEF) was calculated using the formula EF% = ([LV end-diastolic volume - LV end-systolic volume]/LV end-diastolic volume) x 100. Fractional shortening (FS) was calculated as FS%= ([LV end diastolic dimension LV end-systolic dimension]/LV end-diastolic dimension) x 100. Ten weeks following study start all animals were sacrificed and heart, quadricep and liver were taken for biochemical and histological analyses.

8.1.3. Assessment of cardiac function by ultrasound

[0188] Cardiac function was assessed by transthoracic echocardiography using high resolution microimaging systems (Vevo™ F2 LT Preclinical Imaging System, VisualSonics). Briefly, anesthetized spontaneously breathing mice (0.5-3% isoflurane and 98.5-99% O2) were placed in the supine position on a temperature-controlled heating platform to maintain their body temperature at ~37°C. The heating platform also contained electrocardiography (ECG) leads that connect to the animal’s upper and lower paws and measure the animal’s heart rate during recordings. For animals 2 weeks of age, due to their size, copper tape was used to extend the ECG leads to their paws. Nair™ was used to remove hair and expose the skin to the probe. Parasternal short-axis M mode tracings of left ventricle (LV) were recorded for LV mass and LV ejection fraction (EF) calculations. For mice at 2 weeks of age, transducer head UHF71xwas utilized and for mice > 4 weeks of age transducer head UHF46x was utilized. This was due to the size of the heart and frequency required to obtain a clear image. EF was used to determine systolic function, whereas LV mass was used to determine hypertrophy. Vevo™ Lab software was used for analyses.

8.1.4. Ultrasound echocardiography analysis

[0189] Analysis of ultrasound measurements was performed utilizing the Vevo™ Lab software. Trained personnel reviewed multiple recordings acquired in M-Mode (4-5) per animal and the recording with the median score for ejection fraction percentage was then selected to move forward for statistical analysis of all parameters. Recordings were only used if they met the following criteria: papillary muscles are visible and vertically aligned, heart rate between 300-600 bpm, anterior and posterior ventricle walls clearly visible, systole and diastole can be differentiated and clearly seen, and the intraventricular septum (IVS) is not visible. Recordings meeting these criteria were then run using the AutoLV and values for the following parameters were recorded: ejection fraction percentage (EF%), fractional shortening percentage (FS%), left ventricle mass corrected (LVMass cor) left ventricle internal diameter during systole and diastole (LVIDs & LVIDd). Left ventricle mass was additionally calculated as a ratio to bodyweight outside of the Vevo™ Lab software.

8.1.5. Biochemical Assessments

8.1.5.1 Vector Genome Biodistribution via ddPCR

[0190] Distribution of vector genomes in mouse and nonhuman primate (NHP) tissues (including, but not limited to heart, liver, quadriceps, and liver) were analyzed by using droplet digital PCR (ddPCR; Biorad, Hercules, CA, USA). Briefly, DNA was extracted from different mouse/NHP tissues (QiaAMP™ Fast DNA tissue kit, Qiagen, 51404) and diluted to the same concentration across study samples in nuclease- free water (Invitrogen, AM9937). Diluted DNA was then used to generate thousands of water-oil emulsion droplets using manufacturer recommended protocol (Bio-Rad, 1864101). Duplex PCR amplification was then performed within the water-oil emulsion droplets for vector genome and host-species normalization/housekeeping gene (e.g., RPPrpp30) targets using different fluorescent tags. Droplets were then stratified into positive or negative based on fluorescent signal for respective gene targets (QX200, Bio-Rad, 1864003). Concentration of positive droplets as a function of total droplets in ddPCR reaction was used to calculate vector genomes biodistribution per diploid genome. Primer and probe sets used for vector genome biodistribution ddPCR assay detect coding region of the transgene (e.g., BAG3) or regulatory element (e.g., OPRE) within the transgene and the host-RPP30 gene for normalization.

8.1.5.2 Transgene Expression Assessment via RT-ddPCR

[0191] Expression of transgenes in various mouse and nonhuman primate (NHP) tissues (including, but not limited to heart, liver, quadriceps, and liver) were analyzed by Bio-Rad One-Step reverse transcription Droplet Digital™ PCR (RT-ddPCR; Bio-rad, Hercules, CA, USA). Droplet Digital™ PCR uses TaqMan™ technology to generate a fluorescent signal when PCR occurs across a specific target amplicon. Briefly, RNA was extracted from different mouse/NHP tissues (RNeasy kit, Qiagen, 74104) and diluted to the same concentration across study samples in nuclease-free water (Invitrogen, AM9937), followed by DNase treatment (Thermoscientific, AM1907). Diluted RNA was then used to perform RT-ddPCR using the One-step Reverse transcription ddPCR (RT-ddPCR) Advanced kit (Bio-Rad, 1864021) followed by generation of thousands of water-oil emulsion droplets using manufacturer recommended protocol (BioRad, 1864101). Duplex ddPCR amplification was then performed within the water-oil emulsion droplets for transgene and host-species normalization/housekeeping gene (e.g., RPPrpp30) targets using different fluorescent tags. Droplets were then stratified into positive or negative based on fluorescent signal for respective gene targets (QX200, Bio-Rad, 1864003). Concentration of positive droplets as a function of total droplets in ddPCR reaction was used to calculate relative transgene expression levels. Primer and probe sets used for relative transgene expression assay detect transgene (e.g., BAG3) or regulatory element (e.g. OPRE) within the transgene, and the host-RPP30 RNA for normalization.

8.1.5.3 Therapeutic Protein Expression Assessment via ProteinSimple Jess

[0192] Expression of the therapeutic protein in various mouse and nonhuman primate tissues (including, but not limited to heart, liver, quadriceps, and liver) was analyzed by ProteinSimple Jess™ automated Western Blot. Protein lysate was prepared in RIPA buffer (Abeam, ab156034) with Halt protease and phosphatase inhibitor (Thermo Fisher, 78442), and ~10 ng of total protein was loaded into the ProteinSimple Jess™ automated Western Blot apparatus. Proteins were separated using the 12-230kDa Separation Module (ProteinSimple SM-W004). BAG3 was detected using a Rabbit monoclonal Pan BAG3 antibody (Abeam, ab92309) combined with a Goat anti-Rabbit HRP-conjugated secondary antibody (Protein Simple, DM-001). MYBPC3 was detected using a mouse monoclonal antibody (Santa Cruz, sc-137180 (E-7)). Total protein was measured using the Total Protein Detection Module for Chemiluminescence (ProteinSimple, DM-TP01). Peak height for the BAG3 or MYBPC3 signal normalized to Total Protein were used to generate a semi-quantitative readout of relative protein expression between samples. To normalize protein levels using Jess™, a standard curve was generated using recombinant human BAG3 protein (Novus, NBP1 -72276) or recombinant human MYBPC3, as appropriate.

8.1.6. Immunohistochemistry (IHC)

[0193] Immunohistochemistry was performed on the automated BondRX™ platform (Leica) using 5 pm thick sections of formalin-fixed paraffin-embedded mouse tissues mounted on charged slides. All steps were performed at ambient temperature unless otherwise noted. Slides were baked for 30 minutes at 60°C followed by deparaffinization and epitope retrieval using Bond™ Epitope Retrieval Solution ER1 (Leica) for HA-Tag IHC or Bond™ Epitope Retrieval Solution ER2 (Leica) for Human BAG3 IHC. A peroxide block (Leica) and protein block (Dako) were applied prior to HA-Tag antibody (0.2pg/mL, Cell Signaling Technology, 3724S) or Human BAG3 (0.4pg/mL, abeam, ab246225) incubation for 15 minutes. Detection included Anti-HRP Polymer (Leica) with DAB chromogen (Leica) color development and hematoxylin counterstain (Leica). Upon protocol completion, slides were removed from Bond™ RX, dehydrated in a series of graded alcohols and xylenes, and cover slipped with Micromount (Leica). To quantify the fraction of % positive cells in the heart, individual scores were assigned for each heart section for both 10 and 20X magnification fields of view (FOV). These included 2 FOV of the inner left ventricular free wall containing papillary muscle (P1 , P2), 3 FOV of the outer left ventricular free wall (LV1 , LV2, LV3), 2 FOV centered on the interventricular septum (IVS1 , IVS2) and 3 FOV of the right ventricular free wall (RV1 , RV2, RV3). Individual FOV scores and individual and group means were recorded.

8.1.7. Hematoxylin and Eosin (H&E) Stain

[0194] 5 pm thick sections of formalin-fixed paraffin-embedded mouse tissues mounted on charged slides were deparaffinized in xylene and then hydrated through graded alcohols into water. Slides were then put in Carazzi’s hematoxylin, washed in tap water, and placed into 95% ethanol. Then, slides were put in eosin-phloxine solution and ran through graded alcohols to xylene. After, stained slides were cover slipped using Permount.

8.1.8. Masson’s Trichrome Stain

[0195] 5 pm thick sections of formalin-fixed paraffin-embedded mouse tissues mounted to charged slides were deparaffinized in xylene and then hydrated through grade alcohols into water. Bouin's solution was used as a mordant. After Bouin's, the slides were rinsed well before transferring to Weigert's hematoxylin. Next, they were placed in Biebrich scarlet/acid fuchsin, followed by phosphomolybdic acid. The slides were stained with aniline blue before dehydrating with graded alcohol alcohols, clearing with xylene, and cover slipping with Permount.

8.1.9. In situ hybridization (ISH)

[0196] OPRE ISH was completed on heart sections mounted on charged slides using RNAscope™ assay (Advanced Cell Diagnostics) and OPRE probe (ACD, cat#518628) on a BondRX™ autostainer (Leica Microsystems) according to manufacturer guidelines.

[0197] Whole hearts were surveyed at 100x (10x objective) to assess distribution of OPRE ISH staining. 50% of each area (left ventricle, right ventricle, interventricular septum) was evaluated at 200x and assigned to a semiquantitative increment of 5% total OPRE ISH positive cardiomyocytes (final value for each region is between 0-100).

8.2. Example 1 : rAAV with targeting peptides have superior cardiotropism in mice

[0198] WT mice (N=4 per group) received IV injections of (i) a rAAV having modified capsid proteins with a SAQRGDRGQI (SEQ ID NO:1) targeting peptide in VR III and liver-toggle mutations in VR I (alanine at position 267 and threonine at position 269) (VP1 sequence as set forth in SEQ ID NO:8 (“Variant 1”)) and encoding eGFP, (ii) a rAAV having modified capsid proteins with a ENRRGDFNNL (SEQ ID NO:4) targeting peptide (VP1 sequence as set forth in SEQ ID NO:11 (“Variant 4”)) and encoding eGFP, or (ii) a rAAV having wild-type AAV9 capsid proteins (VP1 sequence as set forth in SEQ ID NO:32) expressing eGFP at multiple dose levels. Four weeks post injection immunohistochemistry (IHC) in heart tissue sections show improved eGFP expression with the rAAV with Variant 1 capsid proteins at all doses compared to the rAAV having wild-type AAV9 capsid proteins (FIG. 1 A).

[0199] Expression of the vector derived transgene in mouse tissues were analyzed by Bio-Rad One- Step reverse transcription droplet digital PCR (RT-ddPCR). Copies of the vector derived transgene transcript were detected by a primer/probe set targeted within the coding region. RPP30 transcripts were quantified in a duplex reaction and transgene expression was reported as a percentage of RPP30 expression. The RT-ddPCR results confirmed the improved eGFP expression with the rAAV having Variant 1 capsid proteins at all doses tested compared to the rAAV having wild-type AAV9 capsid proteins (FIG. 1 B).

[0200] Furthermore, the eGFP expression with rAAV having targeting peptides were durable in mouse heart tissues (FIG. 2).

[0201] rAAV having the targeting peptides also enhanced the transcription of GFP DNA in heart tissues by 6.3-21 .7 times and 3.7-69.3 times respectively, compared to the GFP transcription with rAAV having wild-type AAV9 capsid proteins at various doses (FIGS. 3A, 3B and 3C).

8.3. Example 2: rAAV with targeting peptides have superior cardiotropism in NHPs

[0202] Cynomolgus macaques (N=3 per group) received IV injections of rAAV as in Example 1 at 1e14 vg/kg (high dose) or at 1 e13 vg/kg (low dose). Protein expression in the heart was assessed four weeks post injection by HA-IHC and RT-ddPCR.

[0203] Four weeks post injection immunohistochemistry (IHC) in heart tissue sections showed modest improvement in eGFP expression with rAAV having targeting peptides at high dose level compared to rAAV having wild-type AAV9 capsid proteins (FIG. 4). IHC results of liver and DRG tissue sections show liver and DRG detargeting in NHP with the rAAV with Variant 1 capsid proteins compared to rAAV having wild-type AAV9 capsid proteins. At low dose level, eGFP expression was dramatically increased with rAAV having the targeting peptides compared to rAAV having wild-type AAV9 capsid proteins. The increase is more striking at low dose level compared to the high dose level (FIG. 5). The results were confirmed by the percentage GFP+ cells (FIGS. 6A and 6B) and the GFP mRNA detection by ddPCR (FIG. 7A and 7B). The superior transduction of cardiomyocytes of rAAV having capsid proteins with targeting peptides compared to wild-type AAV9 capsid proteins were observed across NHP heart regions, including ventricles, atria, and IVS.

[0204] The results further indicate that rAAV having Variant 1 capsid proteins achieved clinically meaningful and uniform cardiac transduction at 1e13 vg/kg (low dose) across NHP heart regions (FIG. 8).

[0205] Overall, the data show that rAAV having capsid proteins with the targeting peptides have superior cardiac transduction compared to rAAV with wild-type AAV9 capsid proteins in NHPs. 8.4. Example 3: Cardiotropic AAV Vector Encoding BAG3 in a Mouse Model of Myocardial Infarction

[0206] This Example describes a study with a rAAV having a capsid with significantly improved cardiac transduction and robust liver detargeting compared to wild-type AAV9. The study employed a mouse model of myocardial infarction (Ml) with diminished left ventricular (LV) function and employed B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) as a therapeutic transgene.

8.4.1. Materials and Methods

8.4.1.1 Construct Design and Myocardial Infarction Model

[0207] A rAAV vector having modified capsid proteins with a SAQRGDRGQI targeting peptide (SEQ ID NO:1) in VR III and liver-toggle mutations in VR I (alanine at position 267 and threonine at position 269) (VP1 sequence as set forth in SEQ ID NO:8 (“Variant 1”)) and an encapsidated polynucleotide encoding human BAG3 under the control of an MLC2 promoter was designed and produced. The encapsidated nucleic acid is shown schematically in FIG. 9. The encapsidated nucleic acid included, in the 5’ to 3’ direction, a 5’ ITR, MLC-2 promoter, the BAG3 coding sequence, three hemagglutinin (HA) tags, an optimized posttranscriptional regulatory element (OPRE), a bovine growth hormone (BGH) polyadenylation signal sequence, and a 3’ ITR.

[0208] Myocardial infarction (Ml) was surgically induced in mice by a distal ligation of the left anterior descending (LAD) coronary artery creating sustained ischemia and infarction in the LV anterior wall. Cardiac infarction was confirmed by weekly echocardiography. Six weeks following surgery, when Ml mice showed significantly diminished LV function compared to WT control mice (no surgery), Ml mice received IV injection of rAAV at 2e12vg/kg (a dose that led to >70% transduction of the mouse myocardium in unoperated wild type mice in biodistribution studies). Global LV function was evaluated in all mice after light sedation (2% isoflurane) using a Vevo 770 imaging system and a scan head (VisualSonics, Miami, Florida). The left ventricular ejection fraction (LVEF) was calculated using the formula EF% = ([LV end-diastolic volume - LV end-systolic volume]/LV end-diastolic volume) x 100. Fractional shortening (FS) was calculated as FS%= ([LV end diastolic dimension LV end-systolic dimension]/LV end-diastolic dimension) x 100. Ten weeks following study start all animals were sacrificed and heart, quadricep and liver were taken for biochemical and histological analyses. The study timeline is shown schematically in FIG. 10.

8.4.1.2 HA Immunohistochemistry (IHC) Methods

[0209] Protein expression in the heart was assessed 10 weeks post Ml surgery (4 weeks post AAV injection) with IHC using an HA antibody or JESS™ automated western blot (ProteinSimple®, Bio- Techne) using an antibody that recognizes human and mouse BAG3.

[0210] Immunohistochemistry was performed on the automated Bond™-RX platform (Leica) using 5 pm thick sections of formalin-fixed paraffin-embedded mouse tissues mounted on charged slides. All steps were performed at ambient temperature unless otherwise noted. Slides were baked for 30 minutes at 60 °C followed by deparaffinization and epitope retrieval using Bond™ Epitope Retrieval Solution ER1 (Leica). A peroxide block (Leica) and protein block (Dako) were applied prior to HA-Tag antibody (Cell Signaling Technology, 3724S) incubation for 15 minutes. Detection included Anti-HRP Polymer (Leica) with DAB chromogen (Leica) color development and hematoxylin counterstain (Leica). Upon protocol completion, slides were removed from the Bond™-RX, dehydrated in a series of graded alcohols and xylenes, and coverslipped with Micromount™ medium (Leica).

8.4.1.3 Therapeutic Transgene Expression (RT-ddPCR)

[0211] Expression of the vector derived transgene in mouse and NHP tissues was analyzed by Bio-Rad One-Step reverse transcription Droplet Digital™ PCR. Droplet Digital™ PCR uses TaqMan™ technology to generate a fluorescent signal when PCR occurs across a specific target amplicon. PCR reactions are divided into thousands of nano-droplets prior to thermal cycling. The presence of fluorescent signal is used to sort droplets into positive and negative groups. Positive droplets are counted in order to determine the number of template molecules in the original sample. Copies of the vector derived transgene transcript are detected by a primer/probe set targeted within the coding region. RPP30 transcripts are quantified in a duplex reaction and transgene expression is reported as a percentage of RPP30 expression.

8.4.2. Results

[0212] Histological analysis of heart tissues confirmed the presence of a surgically-induced infarct in all Ml mice with no evidence of test article related safety findings in any tissue analyzed (heart, skeletal muscle and liver). Immunohistochemistry demonstrated transduction of 70% of the myocardium and BAG3 expression throughout the heart with little to no expression in liver or skeletal muscle, (see FIGS. 11-13). The rAAV rescued the cardiac functional deficit in Ml mice (FIGS. 14A-14B), with an approximately 40% improvement in LV function by echocardiography. Hearts of mice treated with Variant 1 rAAV was associated with 38% increase in ejection fraction three weeks after the rAAV treatment (FIGS. 14C-14D).

[0213] In summary, the rAAV having Variant 1 capsid proteins demonstrated safety and robust therapeutic efficacy in an animal model of cardiac dysfunction at doses significantly lower than previously reported with a rAAV with wild-type AAV9 capsid proteins (Knezevic et al, 2016, J Am Coll Cardiol Basic Trans Science 1 (7):647-656).

8.5. Example 4: Efficacy of cardiotropic rAAV having Variant 3 capsid proteins in a murine conditional KO model of BAG dilated cardiomyopathy

[0214] Two rAAV having Variant 3 capsid proteins, one encapsulating a polynucleotide comprising an OPRE and one lacking the OPRE, each further comprising BAG3 transgene under the control of the CK8e promoter were designed and produced. Efficacy of constructs with or without OPRE was assessed to determine lowest expression for therapeutic efficacy in a mouse BAG3-cKO model. Mice were injected intravenously (i.v.) with capsids and monitored with ultrasound echocardiography as described in Section 8.1 .4, with the first echocardiography assessment taken before the i.v. injection and later at weeks 2, 4, 6, 10, and 14 post-injection (WPI). Treatment details for each group are provided in Table 1 .

[0215] Ejection fraction percentage at 2 WPI are shown in FIG. 15A. Differences in percentage of fractional shortening associated with each treatment condition are presented in FIG. 15B. Assessment of treatment condition-related changes to left ventricular posterior wall (LVPW) thickness is shown in FIG. 15C. Change in ejection fraction percentage from 0 WPI to 2 WPI is shown in FIG. 15D.

8.6. Example 5: Evaluation of a cardiotropic rAAV having Variant 3 capsid proteins in WT mice and in a conditional KO mouse model of BAG dilated cardiomyopathy

[0216] A rAAV having Variant 3 capsid polypeptides and encapsulating a polynucleotide comprising BAG3-3xHA under the control of the MLC-2 promoter was designed and produced. In a first set of assessments, WT mice were injected intravenously (i.v.) with the rAAV or buffer and biodistribution of vector genome in heart and liver tissues were assessed as described in Sections 8.1 .5.1 at 8 and 28 days WPI. BAG3 RNA expression was assessed as described in Section 8.1 .5.2 at 8 and 28 days WPI. Treatment details for each group of mice in the first set of assessments are provided in Table 2A.

[0217] Vector genome levels in the heart and liver at 8 days WPI are shown in FIGS. 16A-16B and vector genome levels in the heart and liver at 28 days WPI are shown in FIGS. 16C-16D, respectively.

BAG3 RNA levels in hearts and livers at 8 days WPI are shown in FIGS. 16E-16F and BAG3 RNA expression levels in hearts and livers at 28 days WPI are shown in FIGS. 16G-16H, respectively. [0218] In a second set of assessments, cKO mice and control WT mice were injected intravenously (i.v.) with the rAAV or formulation buffer and monitored with ultrasound echocardiography as described in Section 8.1 .4. Biodistribution of vector genome in heart tissues was assessed as described in Section 8.1 .5.1 . BAG 3 expression was assessed as described in Sections 8.1 .5.2 and 8.1 .5.3. Percentage HA- positive cells were determined with IHC as described in Section 8.1 .6. Treatment details for each group of mice in the second set of assessments are provided in Table 2B.

[0219] Ejection fraction percentage at 4 WPI is shown in FIGS. 161 and 16J. Fractional shortening is shown in FIG. 16K. Left ventricular interior diameter and left ventricular posterior wall thickness values are shown in FIGS. 16 L and 16M, respectively.

[0220] BAG3 RNA expression and vector genome levels in hearts of mice are shown in FIGS. 16N and 160, respectively. BAG3 protein expression is shown in FIG. 16P. BAG3 protein levels assessed by HA- IHC of heart tissues are shown in FIG. 16Q. BAG3-HA protein levels as measured by JESS™ are shown in FIG. 16R.

8.7. Example 6: Comparative Evaluation of two cardiotropic rAAV having Variant 3 capsid proteins in WT mice

[0221] Two rAAV having Variant 3 capsid proteins, one encapsulating a polynucleotide encoding BAG3 under the control of the CK8e promoter (see Example 4 in Section 8.5), and the other encapsulating a polynucleotide encoding BAG3 under the control of the MLC-2 promoter (see Example 5 in Section 8.6) were intravenously (i.v.) delivered to WT mice at the dose of 2e13 vg/kg. Eight days WPI, biodistribution of vector genome in heart and liver tissues was assessed as described in Section 8.1.5.1 and BAG 3 RNA expression in heart and liver tissues was assessed as described in Sections 8.1 .5.2.

[0222] Levels of vector genome trended higher in hearts of mice treated with the rAAV whose polynucleotide comprised the CK8e promoter (FIG. 17A). Similarly, levels of vector genome trended higher in livers of mice treated with the rAAV whose polynucleotide comprised the CK8e promoter (FIG. 17B). BAG3 mRNA expression was approximately four times higher in hearts of mice treated with the rAAV whose polynucleotide comprised the CK8e promoter as compared to the MLC2 promoter (FIG. 17C); this effect was not seen in liver (FIG. 17D). 8.8. Example 7: Biodistribution of Vector Genome and Expression of BAG3 in Heart of Mice Treated with Cardiotropic rAAV having Variant 1 Capsid Proteins

[0223] A first set of rAAV comprising Variant 1 capsid proteins and encapsulating polynucleotides comprising a BAG3 coding sequence under the control of MLC-2, aMHC, TNNT2-400 or TNNT2-600 promoters were designed. Each of the rAAV was assessed in WT mice at two dose levels, 2e12 vg/kg and 2e13 vg/kg. Biodistribution of vector genome in heart tissues was assessed as described in Section 8.1 .5.1 . BAG 3 expression was assessed as described in Sections 8.1 .5.2 and 8.1 .5.3. Percentage HA- positive cells were determined by IHC as described in Section 8.1 .6. The results of these assessments are provided in Table 3.

[0224] A second set of rAAV having Variant 1 capsid proteins and encapsulating a polynucleotide comprising a BAG3 coding sequence were designed. The first rAAV (Native 1) comprised a polynucleotide comprising a BAG3 5’UTR in addition to a native BAG3 promoter sequence; the second construct (Native 2) comprised a BAG3 5’ and a 3’ UTR in addition to a native BAG3 promoter sequence; and the third construct (Native 3) comprised a native BAG3 promoter sequence and a BAG3 5’ UTR, a portion of which was deleted. Each rAAV was assessed in WT mice at two dose levels, 2e12 vg/kg and 2e13 vg/kg. Treatment details for each group are provided in Table 4.

[0225] Biodistribution of vector genomes of both the first and second set of rAAV was assessed in heart tissues as described in Section 8.1 .5.1 and BAG 3 RNA expression obtained with the first and second set of rAAV was assessed as described in Section 8.1 .5.2. Protein expression obtained with two of the rAAV of the second set of constructs was assessed as described in Section 8.1 .5.3. Percentage HA-positive cells were determined with IHC as described in Section 8.1 .6.

[0226] BAG3 mRNA expression levels for each treatment group was assessed in the heart at two dose levels, 2e12 vg/kg and 2e13 vg/kg. Overall, the level of BAG3 RNA expression was higher when mice were treated with 2e13 (FIG. 18A). Vector genome levels for the 2e12 vg/kg dose resulted lower levels of vector genome in the heart as compared to the 2e13 vg/kg dose (FIG. 18B). BAG3 protein expression in the heart was assessed for Native 1 and Native 2 rAAV constructs against controls with different amounts of BAG3 relative to baseline level of protein (FIGS. 18C-18D). IHC assessments revealed a higher percentage of HA-positive cells in heart of mice treated with rAAV construct comprising Native 2 when compared to those treated with constructs comprising Native 1 or Native 3 at both dose levels (FIGS. 18E-18G).

8.9. Example 8: Evaluation of Cardiotropic rAAV Comprising Variant 1 Capsid Proteins and Native 2 Polynucleotide in a Mouse Model of Myocardial Infarction

[0227] rAAV comprising Variant 1 capsid polypeptides and encapsulating the “Native 2” polynucleotide as described in Section 8.8 was evaluated in a mouse model of Ml (n=10) as described in Section 8.1.2 against mice that had a sham surgery (n=5). Mice were injected intravenously (i.v.) with 2e13 vg/kg of the rAAV (Ml mice) or formulation buffer (sham mice) and monitored with ultrasound echocardiography as described in Section 8.1 .4, with the first echocardiography assessment taken two weeks prior to the Ml or sham surgery (FIG. 19A).

[0228] Biodistribution of vector genomes was assessed in heart tissues as described in Section 8.1 .5.1 and BAG 3 mRNA expression was assessed as described in Section 8.1 .5.2. BAG3 protein expression in hearts was assessed as described in Section 8.1 .5.3.

[0229] Vector genome and BAG3 mRNA expression are shown in FIGS. 19B and 19C, respectively. FIGS. 19B-19C also include data obtained in WT mice (see Example 5 in Section 8.6), and data from rAAV having Variant 1 capsid proteins and encapsulating polynucleotides having a BAG3 coding sequence operably linked to a MLC2 promoter (see Example 3 in Section 8.4). Protein expression of human BAG3 in hearts of Ml mice is shown in FIG. 19D (Native 2 data is shown for 2e13 vg/kg dose 5 WPI; MLC2 data is shown for 2e12 vg/kg dose 3 WPI).

8.10. Example 9: Evaluation of the Safety and Time Course of Expression of Cardiotropic rAAV Comprising Variant 1 Capsid Proteins and Native 2 Polynucleotide in WT Mice

[0230] A study was conducted to evaluate the safety and time course of expression of rAAV comprising Variant 1 capsid proteins and the “Native 2” polynucleotide at two doses (2e13 vg/kg and 1e14 vg/kg) in WT C57BL/6 mice at 14, 28, and 60 days after i.v. delivery. Briefly, mice (N=4 in all groups) were injected intravenously (i.v.) with either 2e13 vg/kg or 1 e14 vg/kg rAAV or formulation buffer and monitored for the duration of the assessments until tissue collection. Biodistribution of vector genomes was assessed in heart tissues as described in Section 8.1 .5.1 and BAG 3 mRNA expression in heart tissues was assessed as described in Section 8.1 .5.2. BAG3 protein expression in heart tissues was assessed as described in Section 8.1 .5.3. Treatment details for each group are provided in Table 5.

BAG3 mRNA expression is shown in FIG. 20A. BAG3 protein levels are shown in FIG. 20B. Vector genome biodistribution in hearts is shown in FIG. 20C.

8.11. Example 10: Evaluation of Cardiotropic rAAV Comprising Variant 4 Capsid Proteins and Polynucleotides Comprising MLC-2 or Native Promoters

[0231] rAAV having Variant 4 capsid proteins and encapsulating polynucleotides comprising either MLC-2 or Native promoters were designed. rAAV were assessed in WT mice at two dose levels, 2e12 vg/kg and 6.32e11 vg/kg. Biodistribution of vector genome in heart tissues was assessed as described in Section 8.1 .5.1 . Transgene RNA expression in heart tissues was assessed as described in Section 8.1 .5.2. Details of each treatment group are provided in Table 6. Table 6

[0232] Transgene RNA expression in heart tissues is shown in FIG. 21 A. Vector genome biodistribution in heart tissues is shown in FIG. 21 B.

[0233] Comparison of BAG3 RNA expression and vector biodistribution data obtained with rAAV having Variant 4 capsid proteins comprising BAG3 transgene under the control of MLC-2 or Native 2 promoters to their Variant 1 rAAV counterparts for the same dose (2e12 vg/kg) showed similar levels for both measures (FIGS. 21 C and 21 D).

8.12. Example 11 : Evaluation of Cardiotropic rAAV Comprising Variant 2 Capsid Proteins and Polynucleotide Encoding BAG3 under the Control of a MLC-2 Promoter

[0234] A rAAV having Variant 2 capsid proteins and encapsulating a polynucleotide comprising a BAG3 coding sequence under the control of MLC-2 promoter was designed. The construct was assessed in hearts of WT mice at three dose levels, 2e13 vg/kg, 2e12 vg/kg and 6.32e12 vg/kg, 28 days after i.v. delivery of the rAAV. Biodistribution of vector genome in heart tissues was assessed as described in Section 8.1 .5.1 . Transgene RNA expression in heart tissues was assessed as described in Section 8.1 .5.2. Details of each treatment group are provided in Table 7. [0235] BAG3 mRNA expression increased in a dose-dependent manner (FIG. 22A). Similarly, vector genome level correlated with the dose of the rAAV (FIG. 22B).

8.13. Example 12: Comparative Evaluation of Cardiotropic rAAV Comprising Variant 2 and Variant 4 Capsid Proteins in cKO Model of BAG3 Dilated Cardiomyopathy

[0236] A rAAV comprising Variant 2 capsid proteins and a rAAV comprising Variant 4 capsid proteins, each encapsulating a polynucleotide comprising BAG3 under the control of a MLC-2 promoter was assessed in the hearts of BAG3 cKO mice at 2e13 vg/kg with ultrasound electrocardiography as described in Section 8.1 .4 at 2-week intervals. Biodistribution of vector genome in heart tissues was assessed six weeks after treatment as described in Section 8.1.5.1. Transgene RNA expression in heart tissues was assessed six-weeks after treatment as described in Section 8.1 .5.2. Details of each treatment group are provided in Table 8.

[0237] Two weeks after i.v. delivery, ejection fraction percentage values improved with both rAAV, but returned to baseline levels at six weeks after delivery (FIG. 23A). Similarly, fractional shortening percentage values for both constructs increased initially and returned to baseline levels at six weeks after delivery (FIG. 23B). Cardiac structural endpoints (LV mass and wall diameter) showed slower progression to dilation with rAAV (FIGS. 23C-23E).

[0238] Vector genome and BAG3 mRNA levels in heart tissues are shown in FIGS. 23F and 23G, respectively. Both rAAV were associated with an increase in BAG3 protein expression relative to controls; however, BAG3 protein expression was higher in hearts of mice treated with rAAV comprising Variant 4 capsid proteins (FIGS. 23H-23K).

8.14. Example 13: Biodistribution of Vector Genome and Expression of MYBPC3 in Hearts of WT Mice Treated with a Cardiotropic rAAV having Variant 1 Capsid Proteins

[0239] A set of rAAV comprising Variant 1 capsid proteins and encapsulating polynucleotides comprising a MYBPC3 coding sequence under the control of TNNT2, MHCK7, CK8e, and MSEC-725A promoters were designed. Each of the rAAV was assessed in WT mice at two dose levels, 2e12 vg/kg and 2e13 vg/kg. Biodistribution of vector genome in heart and muscle tissues was assessed as described in Section 8.1.5.1. MYPBC3 RNA and protein expression was assessed as described in Sections 8.1.5.2 and 8.1 .5.3, respectively. The treatment groups used in these assessments are provided in Table 9.

[0240] All rAAVs showed high MYBPC3 mRNA expression in mouse heart tissue at the 2e12 vg/kg dose and at the 2e13 vg/kg dose (FIG. 24A). MYBPC3 RNA levels were much lower in quadriceps muscles of mice administered with the same doses (FIG. 24B). At both doses, vector genome DNA levels were higher in the heart than in the quadriceps (FIGS. 24C and 24D, respectively). Next, RNA levels were normalized to vector genome copy numbers to assess relative RNA expression per vector. This assessment revealed that TNNT and MHCK7 promoters were associated with higher levels of mRNA expression per vector DNA in heart than in quadriceps, whereas CK8e and MSEC-725A promoters were associated with equal or lower levels of mRNA expression per vector DNA in heart than in quadriceps (FIGS. 24E and 24F).

[0241] The level of cMYBPC3 protein expression in quadriceps was negligible when measured by JESS™ (data not shown). In heart of mice administered with 2e12 vg/kg rAAV, protein expression of cMYBPC3 was comparable to levels in WT control mice (FIGS. 24G and 24H) despite relatively high RNA expression. cMYBP3 protein expression in hearts of mice treated with 2e13 vg/kg rAAV was higher than cMYBP3 protein expression in hearts of control mice and the highest level of protein expression was associated with the construct comprising CK8e promoter, providing approximately five-fold increase in cMYBPC3 protein expression over wild-type with relatively low variability (FIGS. 24G and 24H). An approximately three-hold increase in cMYBPC3 expression over wild-type was observed with the MSEC- 725A construct; however, a higher degree of inter-sample variability was observed.

8.15. Example 14: Evaluation of the Efficacy of a Cardiotropic rAAV Comprising Variant 1 Capsid Proteins in a KI Model of MYPBC3 Hypertrophic Cardiomyopathy

[0242] The rAAV comprising Variant 1 capsid proteins and encapsulating polynucleotides comprising a MYBPC3 coding sequence under the control of CK8e promoter (see Example 13 in Section 8.14) was evaluated in a KI model of MYPBC3 hypertrophic cardiomyopathy described in Section 8.1 .1 .2. The rAAV construct was administered intravenously (i.v.) to 10-week-old mice at one of the two dose levels, 6.32e12 vg/kg and 2e13 vg/kg. Cardiac function was assessed with ultrasound echocardiography as described in Section 8.1.4, weekly for two weeks before the rAAV administration and 2, 4, 6, 10, 15, 17, and 22 weeks after the rAAV administration. Biodistribution of vector genome in heart and muscle tissues was assessed as described in Section 8.1 .5.1 . MYPBC3 RNA and protein expression was assessed as described in Sections 8.1 .5.2 and 8.1 .5.3, respectively. The treatment groups used in these assessments are provided in Table 10.

[0243] KI Mice treated with the rAAV comprising Variant 1 capsid proteins showed efficacy around IQ-

15 weeks after administration relative to control KI mice; however, ejection fraction percentage was similar between rAAV-administered and control KI mice in later timepoints (FIGS. 25A and 25B).

[0244] Vector genome levels in the heart was correlated with the dose of rAAV that was administered, whereas hearts of mice administered with the rAAV having AAV9 capsid proteins had limited amounts of vector genome (FIG. 25C). Similarly, there was a dose-dependent increase in MYPBC3 RNA levels in hearts of KI mice administered with rAAV having Variant 1 capsid proteins but negligible increase in MYPBC3 RNA levels in hearts of KI mice administered rAAV with AAV9 capsid proteins (FIG. 25D). Among groups of KI mice, MYPBC3 protein level in the heart was highest in the group treated with 2e13 vg/kg rAAV having Variant 1 capsid proteins (FIG. 25E).

8.16. Example 15: Evaluation of MYBPC3 Expression and Safety of Cardiotropic rAAVs Comprising Variant 4 Capsid Proteins in WT Mice

[0245] A set of rAAVs comprising Variant 4 capsid proteins encapsulating polynucleotides comprising a MYBPC3 coding sequence under the control of MYH7, CSRP3, HRC, and MYOZ2 promoters was designed. Each of the four rAAVs was assessed in WT mice at two dose levels, 2e12 vg/kg and 2e13 vg/kg. Biodistribution of vector genome in heart and muscle tissues was assessed 28 days postadministration as described in Section 8.1 .5.1 . MYPBC3 RNA and protein expression was assessed 28 days post-administration as described in Sections 8.1 .5.2 and 8.1 .5.3, respectively. The treatment groups used in these assessments are provided in Table 11 .

[0246] Data obtained with the four rAAVs were compared to those obtained with the four rAAVs of Example 13 in Section 8.14.

[0247] Vector genome DNA was relatively low in hearts of mice treated with 2e12 vg/kg rAAVs comprising Variant 1 capsid proteins or rAAVs comprising Variant 4 capsid proteins (FIG. 26A). At 2e13 vg/kg dose, vector genome DNA levels were higher for both sets of rAAVs (FIG. 26A). MYBPC3 RNA expression was higher in hearts of mice administered with rAAV constructs comprising Variant 1 capsid proteins than in hearts of mice administered with rAAVs comprising Variant 4 capsid proteins at both dose levels (FIG. 26B). Some of the rAAVs were associated with 2-fold or higher levels of MYCBP3 protein expression at the 2e13 vg/kg dose relative to wild-type levels (FIG. 26C).

8.17. Example 16: Evaluation of a Cardiotropic rAAV Comprising Variant 2 Capsid Proteins in a KI Model of MYPBC3 Hypertrophic Cardiomyopathy

[0248] A cardiotropic rAAV comprising Variant 2 capsid proteins and encapsulating polynucleotides comprising a MYBPC3 coding sequence under the control of CK8e promoter was assessed in a mouse KI model of MYPBC3 hypertrophic cardiomyopathy at two dose levels, 2e13 vg/kg and 1e14 vg/kg. Briefly, 5-week-old mice were administered the rAAV construct via i.v. injections. Cardiac function was assessed with ultrasound echocardiography as described in Sections 8.1 .3 and 8.1 .4, just before the rAAV administration and 2, 4, 6, 11 , and 14 weeks after the rAAV administration. Biodistribution of vector genome in heart and muscle tissues was assessed as described in Section 8.1 .5.1 . MYPBC3 mRNA and protein expression was assessed as described in Sections 8.1.5.2 and 8.1.5.3, respectively. Percentage HA-positive cells were determined with IHC as described in Section 8.1 .6. The treatment groups used in these assessments are provided in Table 12.

[0249] Therapeutic efficacy was not observed at either dose at weeks 11 and 14 post-injection, and gender differences were not observed on any measure at 11 weeks post-injection (see, FIGS. 27A-27I).

[0250] There was a dose-dependent increase in vector genome DNA in Kl-mice treated with rAAV at 14 weeks after the injections (FIG. 27J). FIG. 27K shows how the vector genome levels in Kl-mice compare to vector genome levels observed in WT mice 8.5 weeks after they were treated with the same rAAV at the same dose. Similarly, there was a dose-dependent increase in MYBPC3 mRNA in hearts of KI mice 14 weeks after they were treated with rAAV (FIG. 27L). FIG. 27M shows how mRNA levels in Kl-mice compare to mRNA levels observed in hearts of WT mice 8.5 weeks after they have undergone the same treatments. ProteinSimple Jess™ results showed a dose-dependent increase in MYBPC3 protein (FIG. 27N). However, Western blot assessments of MYBPC3 protein did not show a dose-dependency in MYBPC3 protein levels (FIG. 270) when the data were normalized to group of WT mice treated with the formulation buffer (FIG. 27P) or to group of KI mice treated with the formulation buffer (FIG. 27Q).

Further, there was increased HA staining in hearts of WT mice treated with the rAAVs than in KI mice treated with the rAAVs (FIGS. 27R and 27S).

8.18. Example 17: Further Evaluation of a Cardiotropic rAAV Vector Comprising Variant 2 Capsid Proteins on Cardiac Function in a KI Model of MYPBC3 Hypertrophic Cardiomyopathy

[0251] The rAAV comprising Variant 2 capsid proteins and encapsulating a polynucleotide comprising a mMYBPC3 coding sequence under the control of CK8e promoter was designed and assessed in young mice of a mouse KI model of MYPBC3 hypertrophic cardiomyopathy at three dose levels, 2e12 vg/kg, 2e13 vg/kg and 6e13 vg/kg. Briefly, 2-week-old KI mice were administered the rAAV via i.v. injections at the dose assigned. Cardiac function was assessed with ultrasound echocardiography as described in Sections 8.1 .3 and 8.1 .4, just before the rAAV administration and 2, 4, and 6 weeks after the rAAV administration. The treatment groups used in these assessments are provided in Table 13.

[0252] At time points up to six weeks after the injections, percent ejection fraction values in all groups of KI mice treated with different doses of rAAV were similar to the percent ejection fraction observed with the buffer-treated KI mice (FIG. 28A-28B). Similar results were observed for fractional shortening (FIG. 28C), left ventricle internal diameters during systole (LVIDs) (FIG. 28D), left ventricle internal diameters during diastole (LVIDd) (FIG. 28E), and LV mass values that were normalized by body weight (FIGS. 28F and 28G).

8.19. Example 18: Further Evaluation of a Cardiotropic rAAV Comprising Variant 2 Capsid Proteins on Safety and Pharmacological Measures in a KI Model of MYPBC3 Hypertrophic Cardiomyopathy

[0253] The rAAV comprising Variant 2 capsid proteins used in Example 17 in Section 8.17, which encapsulated a polynucleotide comprising a mMYBPC3 coding sequence under the control of CK8e promoter was assessed in young mice of a mouse KI model of MYPBC3 hypertrophic cardiomyopathy at three dose levels, 2e12 vg/kg, 2e13 vg/kg and 6e13 vg/kg. Briefly, 2-week-old mice were administered the rAAV via i.v. injections at the dose assigned. Biodistribution of vector genome in heart was assessed 28 days (4 weeks) and 70 days (10 weeks) after the injections as described in Section 8.1 .5.1 . MYPBC3 RNA and protein expression was assessed 28 and 70 days after the injections as described in Sections 8.1 .5.2 and 8.1 .5.3, respectively. Percentage HA-positive cells were determined by IHC 28 and 70 days after the injections as described in Section 8.1 .6. The treatment groups used in these assessments are provided in Table 14.

[0254] Four weeks after the injections, rAAV-treated and formulation buffer-treated KI mice had lower MYBPC3 RNA levels than WT mice treated with rAAV or formulation buffer (FIG. 29A). Similarly, 10 weeks after the injections, MYBPC3 RNA levels were lower in hearts of KI mice treated with rAAV relative to WT controls (FIG. 29A).

[0255] Levels of vector genome after 28 and 70 days are shown in FIG. 29B. Protein levels of MYBPC3 28 days after the injections are shown in FIGS. 29C-29E.

8.20. Example 19: Evaluation of Cardiotropic rAAVs Comprising Variant 3 Capsid Proteins in a KI Model of MYPBC3 Hypertrophic Cardiomyopathy

[0256] A set of rAAVs comprising Variant 3 capsid proteins and encapsulating polynucleotides comprising a human MYBPC3 (hMYBPC3) coding sequence or a mouse MYBPC3 (mMYBPC3) coding sequence under the control of CK8e promoter was assessed in a mouse KI model of MYPBC3 hypertrophic cardiomyopathy at three dose levels, 6e12 vg/kg, 3e13 vg/kg and 1 e14 vg/kg. Briefly, 2-day old (P2) mice were administered the rAAV via i.v. injections at the dose to which they were assigned. Cardiac function was assessed with ultrasound echocardiography as described in Sections 8.1 .3 and 8.1 .4, just before the rAAV administration and 2, 6, 9 and 14 weeks after the rAAV administration.

Biodistribution of vector genome in heart was assessed as described in Section 8.1 .5.1 . MYPBC3 RNA and protein expression was assessed as described in Sections 8.1 .5.2 and 8.1 .5.3, respectively. The treatment groups used in these assessments are provided in Table 15.

[0257] Administration of rAAVs having Variant 3 capsid proteins at 1 e14 vg/kg was associated with improvement in ejection fraction at two weeks and six weeks after rAAV administration (FIGS. 30A-30C). In contrast, administration of rAAV having AAV9 capsid proteins was not efficacious at either timepoint (FIGS. 30A-30C). Vector genome levels are shown in FIG. 30D. MYBPC3 mRNA levels are shown in FIGS. 30E- 30F). MYPBC3 protein levels are shown in FIG. 30G.

8.21. Example 20: Evaluation of Cardiotropic rAAVs in WT mice and a KI Model of MYPBC3 Hypertrophic Cardiomyopathy

[0258] The rAAVs comprising Variant 3 capsid proteins and encapsulating polynucleotides comprising a human MYBPC3 (hMYBPC3) coding sequence or a mouse MYBPC3 (mMYBPC3) coding sequence under the control of CK8e promoter of Example 19 were further assessed in HOM and HET mice in a mouse KI model of MYPBC3 hypertrophic cardiomyopathy and in WT mice. Briefly, young (2-day old) mice were administered rAAV via i.v. injections at the dose to which they were assigned. Similarly, a group of adult (6-week-old) WT mice were administered 2e13 vg/kg rAAV via i.v. injections. Corrected LVmass was assessed as described in Section 8.1.4. Four weeks after the injections, biodistribution of vector genome was assessed as described in Section 8.1 .5.1 and MYBPC3 mRNA expression was assessed as described in Section 8.1.5.2. The treatment groups of young mice used in these assessments are provided in Table 16.

[0259] Vector genome and human MYBPC3 mRNA levels in hearts of young WT mice (group 1) are shown in FIGS. 31 A and 31 B. FIGS. 31 A and 31 B also show vector genome and MYBPC3 mRNA levels following a 2e13 vg/kg dose to adult WT mice. FIG. 31 C shows LVmass for WT mice and HOM KI mice. Mouse hearts in both WT and HOM KI mice grow substantially between weeks 2 and 6; the observed increase in LVmass within that growth period was ~200% in WT mice.

[0260] Levels of vector genome in the heart displayed dose dependency for the rAAV comprising a human MYBPC3 coding sequence (FIG. 31 D). Human MYBPC3 mRNA and protein expression levels were reduced in hearts of HOM KI mice relative to those of WT and HT KI mice (FIGS. 31 E and 31 F). Expression of mMYBPC3 mRNA is shown in FIG. 31 G. [0261] A dose response in levels of 3x-HA tagged MYBPC protein was observed (FIG. 31 H). Dosedependency was not observed when total MYBPC3 levels were assessed by Jess™ (FIG. 311) (assay detects both endogenous mouse and human MYBPC3), which may indicate regulation of total MYBC3 protein levels or preferential binding of anti-MYBPC3 antibody to the endogenous mouse MYBPC3 protein rather than the human MYBPC3 protein.

8.22. Example 21 : Further Evaluation of Cardiotropic rAAVs in WT mice and a KI Model of MYPBC3 Hypertrophic Cardiomyopathy

[0262] The rAAVs comprising Variant 3 capsid proteins and encapsulating polynucleotides comprising a human MYBPC3 (hMYBPC3) coding sequence or a mouse MYBPC3 (mMYBPC3) coding sequence under the control of CK8e promoter of Example 19 were further assessed in HET mice in a mouse KI model of MYPBC3 hypertrophic cardiomyopathy. Briefly, young (2-day old) mice were administered rAAV via i.v. injections at the dose to which they were assigned. Cardiac function was assessed with ultrasound echocardiography as described in Sections 8.1 .3 and 8.1 .4. The treatment groups of young mice used in these assessments are provided in Table 17.

[0263] Treatments with rAAV having Variant 3 capsid proteins and comprising either a human MYBPC3 coding sequence or a mouse MYBPC3 coding sequence were found to prevent disease progression in HET mice, while treatment with rAAV having AAV9 capsid proteins was not observed to prevent disease progression (FIGS. 32A-32C). FIG. 32B shows data 11 WPI, while FIG. 32C shows data 11 WPI, and 13 WPI.

8.23. Example 22: Evaluation of Cardiotropic rAAVs in WT mice and a KI Model of MYPBC3 Hypertrophic Cardiomyopathy [0264] A set of rAAVs comprising Variant 1 , Variant 2, or Variant 3 capsid proteins and encapsulating polynucleotides comprising an eGFP coding sequence under the control of CK8e promoter was assessed in WT mice and a mouse KI model of MYPBC3 hypertrophic cardiomyopathy. Briefly, 2-week- old mice were administered 2e13 vg/kg rAAV via retroorbital injections. 28 days after the injections, biodistribution of vector genome was assessed as described in Section 8.1 .5.1 and eGFP RNA expression was assessed as described in Section 8.1 .5.2. The treatment groups used in these assessments are provided in Table 18.

[0265] In general, vector genome levels were higher in hearts of WT mice relative to their KI counterparts (FIG. 33A). eGFP mRNA levels are shown in FIG. 33B. Highest vector genome and mRNA levels were observed for rAAV having Variant 3 capsid proteins.

8.24. Example 23: In Vitro assessment of BAG3 protein production

[0266] In the first part of the assessment, BAG3 or GFP protein production was evaluated in C2C12 mouse myoblast cells that were transduced with rAAV comprising a polynucleotide comprising BAG3 or GFP under the control of MLC-2, CK8e, or CBh promoter. The rAAV used in the first and second parts of the assessment are provided in Table 19.

[0267] BAG3 protein concentration obtained with JESS™ automated western blot and ELISA are shown in FIGS. 34A and 34B, respectively. GFP levels obtained by flow cytometry are shown in FIG. 34C.

[0268] In the second part of the assessment, iPSC-CM cells were transduced with rAAV comprising Variant 3 capsid proteins comprising BAG3 under the control of MLC-2, CK8e, or CBh promoter. Cells transformed with rAAV comprising Ck8e promoter were associated with higher BAG3 protein expression than those transformed with rAAV comprising MLC-2 promoter (FIGS. 34D and 34E).

8.25. Example 24: rAAV with targeting peptides show favorable properties when administered to NHPs

[0269] This Example describes further studies performed to assess rAAV having capsid proteins comprising targeting peptides. rAAV having AAV9 and Myo4E capsid proteins were included as comparators.

8.25.1. Materials and Methods

[0270] Studies were performed with NHPs (cynomolgus macaques) as outlined in Table 20 and Table 21 . NHPs included in the studies had < 1 :5 AAV NAb titer. The cargo for each rAAV was a nucleotide sequence encoding eGFP operably linked to a CAG promoter. rAAV was administered by intravenous injection. Animals were administered an immunosuppression regimen of tacrolimus, dexamethasone, and rapamycin.

[0271] Whole blood was collected prior to dosing at days -28 and 1 , and post-doing on days 8, 15, 22, 28/29. Whole blood was collected in K2EDTA tubes, aliquoted and stored at -80C until DNA isolation was performed. DNA isolation and digital droplet polymerase chain reaction (ddPCR) were performed. To obtain plasma and blood cell pellet samples, whole blood was collected in K2EDTA tubes and centrifuged. The plasma was transferred to a separate tube and the cell pellet was left in the collection tube and designated as cell pellet. For the groups in Table 20, urine samples were collected with pans placed under the animal cage, and frozen at -80 °C until DNA isolation was performed; and for fecal samples, animals were individually isolated until feces produced. Feces was frozen and stored at -80 °C until DNA isolation was performed. For alanine aminotransferase (ALT) and aspartate aminotransferase (AST) measurements, whole blood was collected in lithium heparin tubes prior to dosing on days -28, 1 , and post-dosing on days 8, 15, and 28/29 and analyzed using an Axcel Clinical Chemistry Analyzer.

[0272] For the groups outlined in Table 20, DNA from whole blood was isolated using Qiagen DSP DNA Blood mini kits following the manufacturer’s protocol and stored at -20 °C. ddPCR was performed using primers to CAG.eGFP and normalized to ng of DNA input or mL of blood. Each sample was run in technical triplicate. Normalizations to ng of DNA input were calculated as follows: VGC/ng= eGFP (Copies/pl)* 20/ Input gDNA Loaded(ng). Normalizations to mL of blood, plasma, or urine were calculated as follows: VGC/ml= eGFP (Copies/ul)* Dilution factor * 1000. Normalizations to mg of feces were calculated as follows: VGC/mg = Target gene concentration (cp/pl)*20/(Weight of feces used for extraction - weight of empty tube).

[0273] For the study outlined in Table 21 , DNA from whole blood was isolated using Qiagen DSP DNA Blood mini kits following the Qiagen protocol and stored at -20 °C. ddPCR was performed using primers to CAG.eGFP and normalized mL of blood input. Each sample was run in technical triplicate.

Normalizations to mL of blood were calculated as follows: VGC/ml= eGFP (Copies/ul)* Dilution factor * 1000. DNA from plasma was isolated using Qiagen QIAmp Circulating Nucleic Acid kits following the manufacturer’s protocol and stored at -20 °C. ddPCR was performed using primers to CAG.eGFP and normalized to mL of plasma input. Each sample was run in technical triplicate. Normalizations to mL of plasma were calculated as follows: VGC/ml= eGFP (Copies/ul)* Dilution factor * 1000. DNA from blood cell pellets was isolated using Qiagen DSP DNA Blood mini kits following the manufacturer’s protocol and stored at -20C. ddPCR was performed using primers to CAG.eGFP and normalized to ng of DNA input or mL of blood. Each sample was run in technical triplicate. Normalizations to ng of DNA input were calculated as follows: VGC/ng= eGFP (Copies/ul)* 20/ Input gDNA Loaded(ng).

8.25.2. Results

[0274] Predose timepoints (Days -28, 1) had VGC/mL values below the limit of detection in all 1e14 vg/kg groups in whole blood.

[0275] Predose timepoints (Days -28, 1) had values below the limit of detection in all 3e13 vg/kg groups in whole blood. Day 8 showed similar levels of vector genomes present in the blood in groups dosed with rAAV having AAV9, Variant 1 , and Variant 4 capsid proteins. Groups dosed with rAAV having Variant 2 and Variant 3 capsid proteins showed higher levels of vector genomes present in the blood on day 8. On days 22 and 28/29 VGCs from rAAV having Variant 2 capsid proteins were reduced approximately 30- fold compared to AAV9 and Variant 3.

[0276] In plasma, predose timepoints (Days -28, 1) had values below the limit of detection in all 1e14 vg/kg dose groups. Day 8 showed similar levels of vector genomes present in plasma across all groups. On days 22 and 28/29 VGCs for rAAV having Variant 1 capsid proteins were reduced approximately 20- fold compared to AAV9.

[0277] Predose timepoints (Days -28, 1) had values below the limit of detection in all 3e13 vg/kg dose groups in plasma. On days 22 and 28/29, VGCs for rAAV having Variant 1 and Variant 2 capsid proteins were reduced compared to rAAV with AAV9 and Variant 3 capsid proteins.

[0278] In the blood cell pellet, predose timepoints (Days -28, 1) had values below the limit of detection in all 1e14 vg/kg dose groups. On days 22 and 28/29 VGCs for rAAV having Variant 1 capsid polypeptides were reduced 100-fold compared to AAV9. [0279] Predose timepoints (Days -28, 1) had values below the limit of detection in all 3e13 vg/kg dose groups in blood cell pellets. On day 8, blood cell pellet samples from the group treated with rAAV having Variant 2 capsid proteins had elevated vector genomes compared to other groups, with some biological replicates above the upper limit of quantification. On days 22 and 28/29, VGCs from rAAV having Variant 1 and Variant 2 capsid proteins were reduced compared to rAAV having AAV9 and Variant 3 capsid proteins in the blood cell pellet.

[0280] The amount of vector genome copies circulating in whole blood, circulating in plasma, and found in blood cell pellet are shown in FIGS. 35A-37B.

[0281] In urine from animals in the 1e14 vg/kg dose groups, the predose timepoint (Days -28) had values below the limit of detection in all groups. Days 8 and 15 showed similar levels of vector genomes present in urine across all groups. On days 22 and 28/29 VGCs for rAAV having Variant 1 capsid proteins were elevated compared to AAV9. [0282] In feces from animals in the 1e14 vg/kg dose groups, the predose timepoint (Days -28) had values below the limit of detection in all groups. All timepoints collected showed very few vector genomes in feces, and no significant differences between groups.

[0283] Vector shedding data for urine and feces for the 1 e14 vg/kg dose groups are shown in FIGS. 38A-38B, respectively.

[0284] Liver enzymes (ALT and AST) measured at day 8 for groups administered rAAV at 1e14 vg/kg are shown in FIGS. 39A-39D.

[0285] Vector genome number per diploid genome and payload mRNA expression in cervical DRG are shown in FIG. 40A-40B. Vector genome number per diploid genome and payload mRNA expression in lumbar DRG are shown in FIG. 41 A-41 B. Vector genome number per diploid genome and payload mRNA expression in liver are shown in FIGS. 42A-42B. Percentages of GFP positive liver and DRG cells as measured by IHC are shown in FIGS. 43A-43B.

[0286] Data for individual animals in groups administered rAAV with AAV9, Variant 1 , Variant 2, Variant 3, and Variant 4 capsid proteins are provided in Table 22. Table 22 Table 22

8.26. Example 25: Seropositivity of capsid variants comprising targeting peptides

[0287] The prevalence of antibodies reactive to capsid protein Variants 1-4 was assessed using donor human sera samples using an in vitro transduction assay adapted from Calcedo et al., 2018, Hum Gene Ther Methods 29(2): 86-95, utilizing the capsid of interest expressing a reporter construct (e.g., firefly luciferase expressed under the CMV promoter) to assess antibody neutralization. Briefly, 100 human donor samples approximating the United States demographics of the 2021 US census were purchased from BiolVT (Westbury, NY). Samples were heat inactivated at 56 °C for 30 minutes. Serum samples and vector were incubated for one hour, using an MOI of 1 e4 vector genomes per cell. The serum vector mixture was then transferred onto HEK 293T cells. Serum samples were assayed at a 1 :5 dilution and assayed in technical duplicate. Cells were transduced overnight. Luciferase values were read after 24 hours of transduction using Promega Bright Gio. Samples with average transduction less than 50% of virus alone in a matrix control were considered seropositive. Samples with average transduction greater than 50% of virus alone in a matrix control were considered seronegative. Seronegative and seropositive samples were enumerated and expressed as a percentage of the total number of samples assayed. AAV9 capsid protein was included as comparator. [0288] Results are shown in FIG. 44A (AAV9 capsid protein, Variant 1 capsid protein, and Variant 4) and FIG. 44B (AAV9 capsid protein, Variant 2 capsid protein, and Variant 3 capsid protein). Two sets of donor samples were used, accounting for differences in AAV9 seropositivity reported in FIG. 44A and FIG. 44B.

8.27. Example 26: Evaluation of Cardiotropic rAAVs Having Variant 3 Capsid Proteins in Non-Human Primates

[0289] Two rAAV having Variant 3 capsid proteins, one encapsulating a polynucleotide encoding BAG3 under the control of a CK8e promoter (see Example 4 in Section 8.5), and the other encapsulating a polynucleotide encoding BAG3 under the control of a MLC-2 promoter (see Example 5 in Section 8.6) were intravenously (i.v.) delivered to adult cynomolgus macaques at the assigned doses. Four weeks after the injections, biodistribution of vector genome in heart, quadriceps and liver tissues was assessed as described in Section 8.1.5.1. BAG 3 RNA expression in heart, quadriceps and liver tissues was assessed as described in Sections 8.1.5.2. BAG3 protein expression in heart, quadriceps and liver tissues was assessed as described in Section 8.1 .5.3. Percentage HA-positive cells in heart was determined with IHC as described in Section 8.1 .6. The treatment groups used in these assessments are provided in Table 23.

[0290] Initial results of the vector genome biodistribution and BAG3 RNA expression assessments and BAG3 protein levels obtained with JESS™ automated western blot of the first group are summarized in Table 24 below:

[0291] Results of the IHC assessment of a group 2 animal are shown in FIG. 45. Initial results of the vector genome biodistribution and BAG3 RNA expression assessments and BAG3 protein levels obtained with JESS™ automated western blot of the second group are summarized in Table 25 below: Table 25

[0292] Results of the IHC assessment of two group 3 animals are shown in FIG. 46. Initial results of the vector genome biodistribution and BAG3 RNA expression assessments and BAG3 protein levels obtained with JESS™ automated western blot of the third group are summarized in Table 26 below:

8.28. Example 27: Further Evaluation of Efficacy of Cardiotropic rAAV Having Variant 3 Capsid Proteins in a Murine Conditional KO Model of BAG Dilated Cardiomyopathy

[0293] This Example presents further data obtained from the groups of animals described in Example 4. Ultrasound echocardiography was performed as described in Example 4. Biodistribution of vector genome in heart tissues was assessed as described in Section 8.1 .5.1 . BAG3 expression was assessed as described in Sections 8.1 .5.2. Percentage HA-positive cells were determined by IHC as described in Section 8.1 .6.

[0294] Treatment details for each group and the results of these assessments are provided in Table 27 and FIGS. 47A-47D.

8.29. Example 28: Further Evaluation of Cardiotropic rAAVs Having Variant 3 Capsid Proteins in Non-Human Primates

[0295] Building on the preliminary findings of Example 26 in Section 8.27, the two rAAV having Variant 3 capsid proteins, one encapsulating a polynucleotide encoding BAG3 under the control of a CK8e promoter and the other encapsulating a polynucleotide encoding BAG3 under the control of a MLC-2 promoter were further evaluated in a total four treatment groups of adult cynomolgus macaques. Four weeks after rAAV injections, biodistribution of vector genome in heart, quadriceps and liver tissues was assessed as described in Section 8.1.5.1. BAG3 RNA expression in heart, quadriceps and liver tissues was assessed as described in Sections 8.1.5.2. BAG3 protein expression in heart, quadriceps and liver tissues was assessed as described in Section 8.1 .5.3. Percentage HA-positive cells in heart was determined with IHC as described in Section 8.1 .6. The treatment groups used in these assessments are provided in Table 28.

[0296] Results of the vector genome biodistribution (vg/dg) for all treatment groups are summarized in

Table 29 below:

[0297] Results of the BAG3 RNA expression (% RPPE30) for all treatment groups are summarized in

Table 30 below: Table 30

ND = not determined

[0298] Results of assessments of BAG3 protein levels obtained with JESS™ automated western blot for treatment groups 2-4 are summarized in Table 31 below. Values are reported as fold change BAG3 expression normalized to tissues from naive NHPs. BAG3 protein levels were not determined for treatment group 1 .

[0299] Percentage HA-positive cells determined with IHC assessment for all treatment groups are summarized in Table 32 below:

8.30. Example 29: Comparative Evaluation of Cardiotropic rAAVs Having Variant 3 Capsid Proteins in Non-Human Primates

[0300] An rAAV having Variant 3 capsid protein encapsulating a polynucleotide encoding BAG3 under the control of a CK8e promoter was evaluated in adult cynomolgus macaques at two doses. Three weeks after rAAV injections, biodistribution of vector genome in heart, quadriceps and liver tissues was assessed as described in Section 8.1.5.1. BAG3 RNA expression in heart, quadriceps and liver tissues was assessed as described in Section 8.1.5.2. BAG3 protein expression in heart was assessed as described in Section 8.1.5.3. Percentage of OPRE-positive cells in heart were determined with ISH as described in Sections.1 .9. The treatment groups used in these assessments are provided in Table 33.

[0301] Vector genome levels in the left ventricle correlated with the dose of rAAV that was administered (FIG. 48A). Similarly, vector genome levels in the right ventricle, IVS, and quadriceps tissues correlated with the rAAV dose (FIG. 48B). BAG3 RNA and protein levels in these tissues were also correlated with the dose of rAAV that was administered (FIGS. 48C-48F). ISH data showing percentage of OPRE positive cardiomyocytes by region for individual animals are provided in Table 34.

9. CITATION OF REFERENCES

[0302] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. In the event that there is an inconsistency between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended.

10. SEQUENCE LISTING

Claims

WHAT IS CLAIMED IS:

1 . A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a modified capsid protein having a targeting peptide within variable region VIII (VR VIII) comprising the amino acid sequence X1X2X3RGDX7X8X9X10, where X1X2X3 is ENK, SAQ, ASS, or ENR and X7, Xs, X9, and X10 are independently selected from any amino acid residue; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein or a myosin-binding protein C, cardiac-type (MYBPC3) protein.

2. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a modified capsid protein having a targeting peptide within variable region VIII (VR VIII) comprising the amino acid sequence X1X2X3RGDYTSM (SEQ ID NO:14), X1X2X3RGDRGQI (SEQ ID NO:12), or XiX₂X₃RGDFNNL (SEQ ID NO:13), where Xi, X2, and X3 are independently selected from any amino acid residue; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein or a myosin-binding protein C, cardiac-type (MYBPC3) protein.

3. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a modified capsid protein having a targeting peptide within variable region VIII (VR VIII), wherein the targeting peptide comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:4; and b. a polynucleotide encapsulated by the capsid and comprising a coding sequence encoding a B-cell lymphoma 2 (Bcl-2) associated anthanogene-3 (BAG3) protein or a myosin-binding protein C, cardiac-type (MYBPC3) protein.

4. The rAAV of any one of claims 1 to 3, wherein the modified capsid protein comprises a peptide segment within variable region I (VR I) comprising an amino acid sequence of SEQ ID NO:7, SEQ ID NO:5, or SEQ ID NO:6.

5. The rAAV of any one of claims 1 to 4, wherein the targeting peptide comprises the amino acid sequence of SEQ ID NO:3 and the capsid comprises a VP1 capsid protein having at least 90% sequence identity to SEQ ID NQ:10.

6. The rAAV of claim 5, wherein the capsid comprises a VP1 capsid protein having at least 95% sequence identity to SEQ ID NO: 10.

7. The rAAV of claim 5, wherein the capsid comprises a VP1 capsid protein having 100% sequence identity to SEQ ID NQ:10.

8. The rAAV of any one of claims 5 to 7, wherein the capsid comprises a VP2 capsid protein having 100% sequence identity to amino acids 138 to 743 of SEQ ID NO:10.

9. The rAAV of any one of claims 5 to 8, wherein the capsid comprises a VP3 capsid protein having 100% sequence identity to amino acids 203 to 743 of SEQ ID NO:10.

10. The rAAV of any one of claims 1 to 4, wherein the targeting peptide comprises the amino acid sequence of SEQ ID NO:1 and the capsid comprises a VP1 capsid protein having at least 90% sequence identity to SEQ ID NO:8.

11 . The rAAV of any one of claims 1 to 4, wherein the targeting peptide comprises the amino acid sequence of SEQ ID NO:2 and the capsid comprises a VP1 capsid protein having at least 90% sequence identity to SEQ ID NO:9.

12. The rAAV of any one of claims 1 to 4, wherein the targeting peptide comprises the amino acid sequence of SEQ ID NO:4 and the capsid comprises a VP1 capsid protein having at least 90% sequence identity to SEQ ID NO:11 .

13. The rAAV of any one of claims 1 to 12, wherein the polynucleotide comprises a coding sequence encoding a BAG3 protein.

14. The rAAV of claim 13, wherein the BAG3 protein is a human BAG3 protein.

15. The rAAV of claim 13 or claim 14, wherein the BAG3 protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NQ:100.

16. The rAAV of claim 13 or claim 14, wherein the BAG3 protein comprises an amino acid sequence having 100% sequence identity to SEQ ID NO: 100.

17. The rAAV of any one of claims 1 to 12, wherein the polynucleotide comprises a coding sequence encoding a MYBPC3 protein.

18. The rAAV of claim 17, wherein the MYBPC3 protein is a human MYBPC3 protein.

19. The rAAV of claim 17 or claim 18, wherein the MYBPC3 protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NQ:200.

20. The rAAV of claim 17 or claim 18, wherein the MYBPC3 protein comprises an amino acid sequence having 100% sequence identity to SEQ ID NQ:200.

21 . The rAAV of any one of claims 1 to 20, wherein the polynucleotide further comprises one or more expression regulatory elements operably linked to the coding sequence.

22. The rAAV of claim 21 , wherein the one or more expression regulatory elements comprise a promoter.

23. The rAAV of claim 22, wherein the promoter is a muscle-specific promoter.

24. The rAAV of claim 22 or claim 23, wherein the promoter is a CK8e promoter, myosin light chain 2 (MLC-2) promoter, BAG3 promoter, MSEC-725a promoter, CK7 promoter, MHCK7 promoter, MYH7 promoter, CSRP3 promoter, HRC promoter, MYOZ2 promoter, TNNT2 promoter, or aMHC promoter.

25. The rAAV of any one of claims 21 to 24, wherein the one or more expression regulatory elements comprises a posttranscriptional regulatory element sequence.

26. The rAAV of claim 25, wherein the posttranscriptional regulatory element sequence comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, at least 99%, or 100% sequence identity to SEQ ID N0:400.

27. The rAAV of any one of claims 21 to 26, wherein the polynucleotide further comprises a polyadenylation signal sequence.

28. The rAAV of claim 27, wherein the polyadenylation signal sequence comprises a bovine growth hormone (BGH) polyadenylation signal sequence.

29. The rAAV of any one of claims 1 to 28, wherein the polynucleotide further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, optionally wherein the 5’ ITR comprises a nucleotide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NG:600, SEQ ID NQ:601 , SEQ ID NQ:602, or SEQ ID NQ:603, and/or wherein the 3’ ITR comprises a nucleotide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NQ:604 or SEQ ID NQ:605.

30. The rAAV of any one of claims 1 to 12, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:810.

31 . The rAAV of any one of claims 1 to 12, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:910.

32. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:810.

33. A polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NQ:910.

34. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid; and b. a polynucleotide according to claim 32 or claim 33 encapsulated by the capsid.

35. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:10, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a nucleotide sequence as set forth in SEQ ID NO:810.

36. A recombinant adeno-associated virus (rAAV) comprising: a. a capsid comprising a VP1 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:10, and VP2 and VP3 portions thereof; and b. a polynucleotide encapsulated by the capsid and comprising a nucleotide sequence as set forth in SEQ ID NQ:910.

37. A pharmaceutical composition comprising the rAAV of any one of claims 1 to 31 and 34 to 36 and a pharmaceutically acceptable excipient.

38. A unit dose comprising the pharmaceutical composition of claim 37.

39. A host cell comprising the polynucleotide of claim 32 or claim 33.

40. A host cell engineered to produce the rAAV of any one of claims 1 to 31 and 34 to 36.

41 . A method of transferring a polynucleotide to the heart of a subject comprising administering the rAAV of any one of claims 1 to 31 and 34 to 36, the pharmaceutical composition of claim 37 or the unit dose of claim 38 to the subject.

42. A method of treating a subject having or at risk of a cardiac disease comprising administering a therapeutically effective amount of the rAAV of any one of claims 1 to 31 and 34 to 36, the pharmaceutical composition of claim 37 or the unit dose of claim 38 to the subject.

43. The method of claim 42, wherein the subject has a cardiac disease (e.g., due to a BAG3 or MYBPC3 mutation).

44. The method of claim 42, wherein the subject is at risk of a cardiac disease (e.g., due to a BAG3 or MYBPC3 mutation).

45. The method of any one of claims 42 to 44, wherein the cardiac disease is a genetic cardiomyopathy, dilated cardiomyopathy (DCM) (e.g., idiopathic DCM, familial DCM, or non-familial DCM), hypertrophic cardiomyopathy, non-ischemic cardiomyopathy, heart failure (e.g., congestive heart failure, heart failure due to reduced ejection fraction, heart failure due to coronary artery disease, acute heart failure, or end-stage heart failure), restrictive cardiomyopathy, left-ventricular non-compaction, atherosclerosis, coronary artery disease, ischemic heart disease, myocarditis, hypertensive heart disease, valvular disease, congenital heart disease, or myocardial infarction.

46. The method of any one of claims 41 to 45, wherein the rAAV, pharmaceutical composition or unit dose is administered systemically.

47. The method of claim 46, wherein the rAAV, pharmaceutical composition or unit dose is administered intravenously.

48. The method of any one of claims 41 to 47, which comprises administering a 6e11 vg/kg to 1e14 vg/kg dose of rAAV to the subject, e.g., 6e11 vg/kg, 1e12 vg/kg, 2e12 vg/kg, 1 e13 vg/kg, 1.5e13 vg/kg, or 1 e14 vg/kg.

49. The method of any one of claims 41 to 47, which comprises administering a 1 e12 vg/kg to 1e14 vg/kg dose of rAAV to the subject.

50. The method of any one of claims 41 to 47, which comprises administering a 1.5e12 vg/kg to 1 ,5e13 vg/kg dose of rAAV to the subject, e.g., 1e13 vg/kg.

51 . The method of any one of claims 41 to 47, which comprises administering a 1 e13 vg/kg to 1e14 vg/kg dose of rAAV to the subject, e.g., 3e13 vg/kg.

52. The rAAV of any one of claims 1 to 31 and 34 to 36, the pharmaceutical composition of claim 37, or the unit dose of claim 38 for use in a method of transferring a polynucleotide to the heart of a subject, optionally wherein the subject has or is at risk of a cardiac disease (e.g., due to a BAG3 or MYBPC3 mutation).

53. The rAAV of any one of claims 1 to 31 and 34 to 36, the pharmaceutical composition of claim 37, or the unit dose of claim 38 for use in a method of treating a subject having or at risk of a cardiac disease (e.g., due to a BAG3 or MYBPC3 mutation).

54. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to claim 52 or claim 53, wherein the cardiac disease is a genetic cardiomyopathy, dilated cardiomyopathy (DCM) (e.g., idiopathic DCM, familial DCM, or non-familial DCM), hypertrophic cardiomyopathy, nonischemic cardiomyopathy, heart failure (e.g., congestive heart failure, heart failure due to reduced ejection fraction, heart failure due to coronary artery disease, acute heart failure, or end-stage heart failure), restrictive cardiomyopathy, left-ventricular non-compaction, atherosclerosis, coronary artery disease, ischemic heart disease, myocarditis, hypertensive heart disease, valvular disease, congenital heart disease, or myocardial infarction.

55. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of claims 52 to 54, wherein the method comprises administering the rAAV, pharmaceutical composition or unit dose systemically (e.g., intravenously).

56. The rAAV for use, pharmaceutical composition for use, or unit dose for use according to any one of claims 52 to 55, wherein the method comprises administering a 6e11 vg/kg to 1e14 vg/kg dose of rAAV to the subject, e.g.,6e11 vg/kg, 1e12 vg/kg, 2e12 vg/kg, 1e13vg/kg, 1 ,5e13 vg/kg, or 1e14 vg/kg.