HEK 293 cells were grown in suspension culture and transfected with 3 plasmids to produce a rAAV vector using standard methods known in the art. One of the plasmids comprised Compound A. The HEK 293 cells were harvested, lysed, flocculated, and the resulting lysate was filtered to produce a clarified lysate. The rAAV vector comprising the BAG3 transgene was purified by a series of chromatography and filtration steps and suspended in a formulation comprising: 10 mM Phosphate pH 7.4 with 350 mM NaCI, 2.7 mM KCI, 5% Sorbitol and 0.002% Pluronic.

Table 2. Vector Productivity

Table. 3. Stability

Table 4. Summary of batch attributes

Example 2: Pharmacology

Compound A pharmacology was evaluated in vitro and in vivo. Cell-based assay data indicated that Compound A effectively transduced and induced the synthesis of functional BAG3 protein in wild-type human induced pluripotent stem cell-derived cardiomyocytes (HiPSC-CM). Human BAG3 protein expression was detected in the hearts of wild-type (WT) mice and WT non-human primates (NHPs) following administration of Compound A. Administration of Compound A in the mouse disease model prevented further deterioration of the disease process. Specifically, it stabilized cardiac structure and function and this correlated with the dose level and amount of human BAG3 protein expression in the heart. Compound A vector transduction and human BAG3 protein expression were evaluated in NHPs. Compound A -treated NHPs had sustained human BAG3 expression in their hearts at the predicted efficacious therapeutic target levels.

Table 5.

Primary Pharmacology In Vitro

Compound A was tested in wild-type human induced pluripotent stem cell derived cardiomyocytes (HiPSC-CM, CDI iCell Cardiomyocyte) to demonstrate transduction (viral genome counts, VGC) and transgene expression (mRNA, protein) as well as stabilization of a BAG3 partner protein (HSPB8). HiPSC-CM were used with the goal of implementing a potency assay in a cell type in which the cardiac troponin promoter element is active, thus allowing expression of the transgene. HSPB8 is a BAG3 partner protein whose stability is dependent on the presence of functional BAG3 and is being used as a surrogate for BAG3 activity (Fang et al., JCI Insight 4(4): e126464 (2019); Fang et al., J. Clin. Invest. 127(8): 3189-200 (2017); Judge et al., JCI Insight 2(14): e94623 (2017)).

HiPSC-CMs were transduced with Compound A and 4 days post transduction transgene mRNA was readily detected (FIG.1). Total BAG3 protein (human endogenous + human transgene) was measured using the Protein Simple WES platform. Total BAG3 protein levels demonstrated a dose responsive increase with increasing vector multiplicity of infection (MOI).

HiPSC-CMs were first transfected with an siRNA targeting endogenous BAG3 expression, which resulted in significant knockdown of endogenous BAG3 protein expression and HSPB8 destabilization (FIG. 2). Subsequently the HiPSC-CMs were transduced with Compound A , resulting in a robust upregulation of total BAG3 protein (human endogenous plus human transgene) and stabilization of HSPB8 partner protein. Primary Pharmacology In Vivo

Primary pharmacology in vivo was investigated in wild-type mice (biodistribution & expression), in a mouse model of disease (characterization of model and evaluation of dose response associated efficacy) and in non-human primates (biodistribution and expression).

Evaluation of biodistribution & expression in wild-type mice

Wild type mice (n:12, age: 8 weeks) were dosed with Compound A (3E13 vg/kg) with a planned necropsy at 3- and 8-weeks post dosing. No significant differences in body weight were detected during the study between Compound A-treated mice and vehicle-treated control mice (FIG. 3A).

Viral genome copies were readily detected in the hearts of Compound A-treated mice at 3 and 8 weeks post-dose (FIG. 3B). At the 8-week timepoint, liver viral genome levels were approximately 175x higher than in the heart. Transgene mRNA expression was highest in the heart with a similar level at both timepoints. In the liver, transgene mRNA expression was approximately 40-50% of the signal in the heart (FIG. 3C). Transgene protein expression (assayed using an antibody specific for human BAG3 protein) was readily detected in the hearts of Compound A-treated mice while no signal was detected in the skeletal muscle and a low signal was detected in the liver (FIG. 3D). When quantifying total BAG3 protein in the heart a similar level of BAG3 expression was detected in hearts of Compound A-treated mice as that detected in control vehicle-treated mice.

Cardiac function (left ventricular ejection fraction) and structure (left ventricular end diastolic volume) were measured by echocardiography at 4- and 7-weeks post dosing. No significant differences were detected in dosed compared to vehicle-treated control mice (FIG. 3E). Finally, the proportion of area in the heart expressing the human transgene was assayed using BAG3 immunohistochemistry showing similar biodistribution at 3- and 8-weeks (74% and 56%, respectively) (FIG. 3F).

Example 3: Characterization of the cKO mouse model of disease:

Mice with cardiac specific knockdown of BAG3 (BAG3 cKO, n:20, male and female) (Fang et al., J. Clin. Invest. 127(8): 3189-200 (2017)) and wild-type control mice (BAG3 cWT, n:20, male and female) were characterized longitudinally using cardiac echocardiography between the ages of 7- and 21- weeks (FIG. 4A-4B). A small but significant deficit in ejection fraction was detected as early as 7-8 weeks of age (BAG3 cKO: 64.2 ± 6.0% vs. BAG3 cWT: 70.5 ± 3.9%) which became larger by 18-21 weeks of age (BAG3 cKO: 31 .9 ± 8.7% vs. BAG3 cWT: 72.2 ± 3.6%). Similarly, a significant increase in left ventricular end diastolic volume was observed in BAG3 cKO mice as compared to wildtype control at 18-21 weeks of age (BAG3 cKO: 93.4 ± 30.4% vs. BAG3 cWT: 61 .5 ± 9.0%). No significant difference in body weight was detected between the two cohorts between 11- 15 and 21-23 weeks of age (FIG. 4C).

As expected, BAG3 protein (measured by Protein Simple WES assay) was absent from the hearts of cKO mice whereas it was readily detected in hearts of control wild-type mice (FIG. 4D). A significant decrease in HSPB8 protein was observed in the hearts of BAG3 cKO mice with the signal being approximately 20% of that measured in wild-type control mice (FIG. 4E). BAG3 protein levels in liver and skeletal muscle tissue of BAG3 cKO mice were the same as in wild-type mice.

Expression levels (mRNA) of heart failure (Nppa, Nppb, Myh7/Myh6 ratio) and fibrosis (Col1a1 , Col1a2, Fn1 , Postn, Timpl) biomarkers were significantly upregulated in the hearts of BAG3 cKO mice compared to wild-type control mice (FIG. 4F-4G). Finally, based on the histological assessment (PicroSirius red staining) of heart tissue, a significantly higher percentage of fibrosis was observed in the hearts of BAG3 cKO as compared to control wildtype mice (FIG. 4H).

Example 4: Evaluation of dose response associated efficacy in mouse model of disease:

To determine whether restoration of BAG3 expression in the heart via AAV gene delivery results in a therapeutic effect, a dose response efficacy study in the cKO mouse model of disease was pursued. BAG3 cKO mice were administered (age: 12 weeks, IV) Compound A at 1 E13 vg/kg (n:15), 3E13 vg/kg (n:15), 9E13 vg/kg (n:15). The study also included two control groups: BAG3 cKO untreated mice (n:20), wild-type untreated mice (n:20). Two weeks prior to treatment, mice were characterized using echocardiography to determine baseline cardiac function and structure. Mice were randomized into groups based on echocardiography readouts (ejection fraction, left ventricular end diastolic volume), body weight, and gender.

Cardiac function (ejection fraction) and structure (left ventricular end diastolic volume) were measured longitudinally using echocardiography (FIG. 5A-B) at 4-, 8-, and 12- weeks post dosing. At week 12, a significant improvement in ejection fraction was detected in mice treated with Compound A at 3E13 vg/kg (42.5 ± 11 .4%) and 9E13 vg/kg (47.9 ± 7.3%) as compared to untreated BAG3 cKO mice (28.2 ± 10.0%). At week 12, a significant improvement in left ventricular end diastolic volume was detected in mice treated with Compound A at 3E13 vg/kg (78.5 ± 12.2uL) and 9E13 vg/kg (71.7 ± 9.0%) as compared to untreated BAG3 cKO mice (97.2 ± 32.5uL). No significant differences in body weight were detected in these mouse groups (FIG. 5C).

Viral genome biodistribution was examined in heart, liver and skeletal muscle tissue collected at necropsy from mice treated with Compound A at 1 E13 vg/kg, 3E13 vg/kg, and 9E13 vg/kg (FIG. 5D). A clear dose response in biodistribution was detected in the heart: 1 E13 vg/kg (0.03 ± 0.01 vg/mTFRC), 3E13 vg/kg (0.19 ± 0.1 vg/mTFRC), 9E13 vg/kg (0.6 ± 0.2 vg/mTFRC). The highest viral genomes were detected in liver (40-45x higher than the heart), with lower vector genomes observed in skeletal muscle (0.2x of the heart level). Transgene mRNA expression was highest in the heart, compared to other tissues examined, with the liver having approximately O.lx the heart level and skeletal muscle having approximately 0.01 x the heart level (FIG. 5E).

Total BAG3 protein expression (human plus mouse) was determined using a Protein Simple WES assay. BAG3 protein was not detected in the hearts of untreated control cKO mice. A dose-responsive increase in BAG3 protein levels was observed in cKO mouse hearts after treatment with Compound A. At the highest dose, approximately 80% (0.78 ± 0.45) of the WT level of BAG3 was detected and at the mid-dose, approximately 20% (0.20 ± 0.11%) of WT BAG3 levels was observed (FIG. 5F). Compound A also induced a dose- responsive increase in HSPB8 protein expression in the same heart tissue samples: approximately 70% (0.68 ± 0.20) at the highest dose, and approximately 35% (0.34 ± 0.06%) of wild-type levels at the mid-dose (FIG. 5G).

Human BAG3 protein expression was quantified using an LC/MS assay specific for the human sequence: at 9E13 vg/kg, the expression was approximately 70% of the level in wild-type mouse heart and, at 3E13 vg/kg, it was approximately 20% of the level expressed in the wild-type mouse heart (FIG. 5H). Transgene spatial expression was determined using in situ hybridization (human BAG3 mRNA) and immunohistochemistry (BAG3 protein, antibody specific for human protein) (FIG. 5I-5J). At a dose of 9E13 vg/kg, 52 ± 18% of cardiomyocytes were positive for human BAG3 mRNA and 52 ± 14% of the heart region was positive for BAG3 protein. At a dose of 3E13 vg/kg, 39 ± 26% of card io myocytes were positive for transgene mRNA and 29 ± 15% of the heart region was positive for transgene protein.

Biomarkers of heart failure and fibrosis were examined using RT-PCR (FIG. 5K). When comparing to untreated control mice, treatment with Compound A at 9E13 and 3E13 vg/kg resulted in a significant decrease in Nppa, Nppb, and the Myh7/Myh6 ratio. A significant decrease was also detected in the levels of Col1 a1 and Postn expression although no significant change was detected in Col1a2, Fn, and Timpl levels. No significant difference following treatment was detected in NT-proBNP levels measured in serum.

Example 5: Evaluation of biodistribution & expression in non-human primates:

To determine the degree of viral genome biodistribution in non-human primates, a cohort of animals was treated with Compound A at 3E13 and 1 E14. Repeated titering after administration led to a more accurate titer of the administered vector and the actual dose levels are 4E13 and 1 .3E14 vg/kg. Actual dose levels were used for modeling and dose projection in Example 8. Each dose level was administered to 1 male and 1 female adult animal. The study also included a pair of vehicle-treated animals. Vehicle consisted of 0.002% pluronic F68 in 10 mM phosphate, 350 mM NaCI, 2.7 mM KCI, and 5% sorbitol in sterile water for injection (pH 7.4). The in-life component of the study was 6 weeks long.

The number of viral genomes detected in the heart increased with vector dose: 0.86 ± 0.15 vg/MfTFRC (4E13 vg/kg) and 2.72 vg/MfTFRC (1 .3E14 vg/kg) (FIG. 6A). Viral genomes in the liver were about 167-243x higher than those in the heart. Viral genomes were also detected in the dorsal root ganglia (0.12-0.35x heart levels), the spinal cord (0.06x heart levels), skeletal muscle (0.2-0.35x heart levels) and testis or ovary (0.04-0.08x heart levels). One female animal administered with the 1 .3E14 vg/kg dose exhibited very low biodistribution and it was determined that this animal had seroconverted prior to dosing and carried neutralizing antibodies against AAV9.

Transgene mRNA expression levels were quantified and normalized either to endogenous HPRT or endogenous BAG3 (FIG. 6B-6C). Expression levels achieved in the heart were approximately 6-8x higher in animals treated with 1 .3E14 vg/kg, compared to the 4E13 vg/kg dose. Transgene mRNA expression in the liver was higher than that detected in the heart: 1 ,8x at the 4E13 vg/kg dose and 6.4x at the 1 .3E14 vg/kg dose. T ransgene expression in the dorsal root ganglia was approximately 0.44x compared to the signal detected in the heart at the 1 .3E14 vg/kg dose. A much lower signal was detected in the spinal cord and skeletal muscle. Upon normalization of transgenic BAG3 mRNA levels to endogenous BAG3 levels, it was found that in the heart, at the 4E13 vg/kg dose level, the transgene mRNA levels were approximately 0.1x that of endogenous BAG3, and 0.8x at the 1 .3E14 vg/kg dose level. In the liver, the signal was higher: 3x endogenous at the 4E13 vg/kg and 25x endogenous at the 1 .3E14 vg/kg.

Human BAG3 protein expression was measured using LC/MS (FIG. 6E-6F). A peptide with a sequence conserved in both human and cyno BAG3 protein was used to measure total protein, whereas a peptide with a sequence specific for the human protein was used to quantify human protein. When comparing BAG3 levels in the heart, 369 ng/mg of protein at the 1.3E14 vg/kg dose, 248 ng/mg of protein at the 4E13 vg/kg dose, and 189 ng/mg of protein in vehicle-dosed animals was detected.

In non-cardiac tissues of vehicle-dosed animals, endogenous BAG3 levels were highest in the spinal cord (157 ng/mg) and lowest in the liver (7 ng/mg). Human transgene protein was only detected in Compound A-dosed animals. At the 4E13 vg/kg dose, approximately 13.9 ng/mg (male animal) and 4.6 ng/mg (female animal) of BAG3 protein was detected in the heart while no transgene protein was detected in liver, dorsal root ganglia, spinal cord, or skeletal muscle. At the 1.3E14 vg/kg dose, approximately 70 ng/mg of BAG3 protein was detected in the heart while no transgene protein was detected in dorsal root ganglia, spinal cord, or skeletal muscle. A small signal (approximately 3 ng/mg) was detected in the liver. When comparing to total BAG3 levels, the human BAG3 protein level detected in the heart was approximately 4% of the total BAG3 level at the 4E13 vg/kg dose level and approximately 30% of total BAG3 at 1 .3E14 vg/kg.

Spatial BAG3 expression was evaluated in the heart using in situ hybridization (FIG. 6D). At the 4E13 vg/kg dose30 ± 11 % (female) and 64 ± 19% (male) of cardiomyocytes stained positive by ISH. At the 1 .3E14 vg/kg dose 89 ± 5% of cardiomyocytes stained positive by ISH (male animal; seroconverted female animal was excluded from this analysis).

Example 6: Toxicology and Safety Pharmacology Brief Summary

The nonclinical safety of Compound A was evaluated in studies in both WT and cKO mice (pharmacology) with biodistribution and toxicology endpoints. In addition, a 6-week exploratory toxicology and biodistribution study (ETS) in cynomolgus monkeys was completed. Safety pharmacology endpoints or the potential for insertional mutagenesis were not evaluated in these early studies.

In the WT mouse study, animals were administered 3E13 vg/kg Compound A by IV bolus with a limited biodistribution, clinical pathology, and histopathology endpoint list evaluated at 3 and/or 8 weeks post dose administration. There was target expression in the WT mouse at 3E13 vg/kg with no toxicity findings.

In the cKO mouse study (1 E13, 3E13, and 9E13 vg/kg Compound A), histopathology findings were limited to animals at the high dose evaluated 12 weeks following dose administration. The Compound A-related findings at the high dose (9E13 vg/kg) consisted of mild cardiomyocyte degeneration/necrosis, however, they were not associated with any changes in cardiac biomarker endpoints of toxicity and occurred in the presence of functional efficacy (/.e., echocardiographic improvement). Functional improvement in the disease mouse model was identified at >3E13 vg/kg identifying the mouse efficacious range.

In the 6-week monkey ETS, Compound A was tolerated at both dose levels evaluated (3E13 and 1 E14 vg/kg) with no Compound A-related microscopic findings. Repeated titering after administration led to a more accurate titer of the administered vector and the actual dose levels are 4E13 and 1.3E14 vg/kg. Based on transient clinical pathology changes while in the presence of a daily immunosuppression regimen (increases in ALT and GLDH, >4E13 vg/kg; increase troponin at 4E13 vg/kg), cytokine increase (TNF at 1 .3E14 vg/kg), the heart, liver, and immune system may be potential target organs with higher dose and/or longer duration.

In summary, the early nonclinical toxicology assessment in mice (WT and cKO) and monkeys provided an adequate safety profile for continued development and supported dosage selection for evaluation in regulatory toxicology.

Toxicology Species Selection

Cynomolgus monkeys (cyno) were selected as a relevant toxicology species for Compound A evaluation. The cynomolgus monkey was selected based on the following weight of evidence: (i) high target homology, (ii) ability to support transgene expression, (iii) demonstrated transgene protein localization at site of action, and (iv) species sensitivity for gene therapy-related toxicity assessment such as acute liver toxicity and microscopic changes in the dorsal root ganglia.

BAG3 has approximately 97% identity across the full-length protein sequence for cynomolgus monkey and human. Moreover, the substantial similarity between cyno and human BAG3 indicates that it is likely to maintain all of the relevant binding partners and interactions. BAG3 binds partner proteins mainly through its WW1 , WW2, IPV1 , IPV2, PXXP, and BAG domains. Comparing cyno and human BAG3 protein sequences, the WW1 , IPV1 , IPV2, and BAG domains have identical sequences while the WW2 and PXXP domains have 97% and 92% identity. Further support comes from the cyno ETS study results that showed significant and dose responsive human BAG3 expression by in situ hybridization (BAG3 mRNA) in heart tissue with digital imaging analysis. Importantly, the human BAG3 expression in the cyno translated to detectable and dose responsive human BAG3 protein levels in the heart.

Furthermore, LCMS evaluation of purified sarcomeres isolated from the cyno ETS hearts (3 heart sections/animal) identified the single peptide unique to the human isoform. This peptide displayed a dose-dependent increase in signal across the two dose levels of the administered human BAG3 transgene (FIG. 7) and supports localization of human BAG3 at the site of sarcomere action. Finally, the human BAG3 transgene was found to rescue the cardiac function deficit in BAG3 cKO mice despite the sequence differences between human and mouse BAG3 (84% identity). Taken together, BAG3 homology, expression, localization, along with species sensitivity for gene therapy-related toxicity assessment support relevance of the cyno as a toxicology species.

Three exploratory toxicity (eTox) studies performed

1. Exploratory WT mouse biodistribution and tolerability study at 3 and 8 weeks following single IV bolus of3E13 vg/kg Compound A.

A biodistribution and toxicology study used male WT mice (BL6/J; 8 weeks old) that were administered 3E13 vg/kg Compound A intravenously via tail vein injection and necropsied following 3- or 8-weeks post injection. A separate control group received a single saline administration. Endpoint evaluations for heart, liver, and skeletal muscle included molecular biology (VGC, RNA, protein; as shown in FIG. 3B-3D), clinical pathology, histopathology, in addition to IHC (protein; FIG. 3F; FIG. 8) and ISH (RNA; FIG.9). The targeted panel of clinical pathology parameters consisted of liver, cardiac and skeletal muscle biomarkers (ALT, AST, ALP, total bilirubin, GLDH, Cardiac Troponin (cTNI), Fatty acid binding protein 3 (FABP3), Myosin light chain 3 (Myl3), and Skeletal troponin I (sTnl)). Animals were evaluated for potential Compound A-related microscopic tissue effects at 8- weeks post dose administration (heart, liver, skeletal muscle, brain, adrenal gland, kidney, lung, spinal cord, and dorsal root ganglia [DRG]).

Table 6

All mice survived to the scheduled sacrifice at 3 or 8 weeks with no Compound A- related changes in body weight (FIG. 3A), clinical pathology markers of heart (cTnl, Myl3, FABP3), liver (ALT, AST, ALP, total bilirubin, GLDH) or skeletal muscle injury (sTNI, Myl3, FABP3) compared to controls. There were no Compound A-related histopathology findings noted at 8-weeks.

Human BAG3 protein immunoreactivity was evaluated by IHC in the heart, liver, and skeletal muscle. IHC revealed a widespread expression pattern that varied in intensity across animals dosed with 3E13 vg/kg Compound A. The BAG3 heart expression pattern and intensity was generally similar at 3- or 8-weeks with heart area staining (FIG 3F; FIG. 8). The left ventricular free wall and interventricular septum of the heart showed slightly greater intensity compared with other locations. Very low expression of BAG3 protein via IHC was noted in liver of all animals in the centrilobular zones as well as marginal expression in the DRG. No BAG3 expression was observed in the skeletal muscle.

Human BAG3 ISH (mRNA) was completed on heart, liver, skeletal muscle from select animals and DRGs from all animals at 8-week time point. Representative images are shown in FIG. 9. ISH labeling in the heart was nuclear and cytoplasmic, variable and labeled fewer card io myocytes than IHC. In the liver, ISH labeling was mostly nuclear and frequently observed in hepatocytes. There was minimal human BAG3 RNA expression in the DRG. Skeletal muscle was BAG3 negative.

In summary, 3E13 vg/kg Compound A administered by the intended route of administration in the clinic, was tolerated in WT mice at 3- and 8-weeks post IV administration. There were no Compound A-related clinical pathology changes in markers of heart, liver, or skeletal muscle injury or microscopic findings. Evaluation by IHC revealed a widespread variable pattern of BAG3 human protein expression in heart of all animals, while there was very low level expression in liver and DRG, and no expression observed in skeletal muscle.

2. Exploratory cKO mouse biodistribution and tolerability study (12 weeks) following single IV bolus of 1E13, 3E13 and 9E13 vg/kg Compound A. An exploratory pharmacology/toxicology study was performed in 12-week old BAG3 cKO dilated cardiomyopathy disease model (DCM) mice treated with a single intravenous tail vein administration of saline or Compound A. The single dose administration of 1 E13, 3E13, or 9E13 vg/kg Compound A was followed by necropsy at 12-weeks post dose. A cohort of cKO mice and a cohort of WT mice were included to establish a baseline for comparison at study start.

Table. 7.

Table 8: Individual animal mortality summary

Mortality occurred across treatment groups including cKO saline control animals (Table 8 above), which was attributed to DCM disease and not related to Compound A treatment. Necropsy was performed at approximately 12-weeks post dosing and serum was collected. No Compound A-related differences in a targeted panel of clinical pathology parameters were observed across any groups (ALT, AST, ALP, GLDH, TBIL, cTNI, MYL3, sTNI, and FABP3). A marker of cardiac toxicity, NT-proBNP (indicative of myocyte stretch) was found to be elevated at 12-weeks post dose in the cKO mouse control group compared to WT mice and indicated the pathological progression of the disease model. There were no Compound A-related changes in NT-proBNP at 12-weeks post dose.

Tissues collected at 12-weeks post dosing included heart, liver, skeletal muscle, spinal cord/DRG, lung, kidney, adrenal gland and brain; however, only heart, liver and skeletal muscle were evaluated for potential histopathology findings. Microscopic heart evaluation of the untreated control cKO mice at baseline (11-12 weeks of age) showed microscopic changes of minimal severity consistent with the DCM disease model (Table 9; severity score compared to WT mice). The saline control cKO hearts at study end (23-25 weeks of age) were similar to the baseline cohort of cKO mouse hearts, except for findings of mild to moderate ventricular dilatation and mild cardiomyocyte atrophy.

In the cKO group treated with 3E13 vg/kg Compound A, signs of efficacy were seen by functional assessment (echocardiography, FIG. 5A and 5B) and by microscopic evaluations (decreases in mean severity of ventricular dilatation and cardiomyocyte atrophy). This dose level (3E13 vg/kg) did not have Compound A-related toxicity findings, although rare highly-expressing karyocytomegalic cardiomyocytes with cytoplasmic basophilia were observed. The cKO group treated with the high-dose (9E13 vg/kg) of Compound A also showed functional improvement (FIG. 5A and 5B) and microscopic signs of efficacy in the heart. While efficacy was still evident at the high dose, there was mild Compound A-related cardiomyocyte degeneration/necrosis with karyocytomegaly, cytoplasmic basophilia and occasionally vacuolation. These microscopic findings in the high dose animals occurred without changes in biomarkers of cardiotoxicity and in the presence of functional (efficacy) indicating continued improvement. As such, clinical doses into this range (/.e., 9E13 vg/kg) would not be precluded.

Immunohistochemistry (IHC) and in situ hybridization (ISH) were used to evaluate the biodistribution of transgene expression in the heart, liver, and skeletal muscle (other tissues including spinal cord/DRG were not evaluated). Human BAG3 protein immunoreactivity by IHC generally increased with dose of Compound A, with variable distribution of signal observed within treatment groups and within individual heart samples (FIG. 10). Digital image analysis indicated that up to a mean of 52% of the heart was positive for BAG3 protein (Table 9; FIG. 5J). Human BAG3 mRNA was detected by ISH and signal was also observed to increase with the dose of Compound A. Digital image analysis indicated up to 52% of the card io myocytes were positive for human BAG3 expression across dose groups (Table 9; FIG. 10; FIG. 5I).

Table 9: Summary of heart microscopic findings, mean human BAG3 percent positive cardiomyocyte protein levels by immunohistochemistry, and mean human BAG3 RNA by in situ hybridization in the hearts of cKO mice.

In summary, evaluation of Compound A in BAG3 cKO mice (1 E13, 3E13, 9E13 vg/kg), administered by the intended route of clinical administration, indicated functional improvement at >3E13 vg/kg as well as dose-dependent expression by ISH (RNA) and IHC (protein) in cardiomyocytes. Evaluation of a targeted set of safety endpoints at 12-weeks post dose did not reveal any Compound A-related clinical pathology changes in markers of liver, heart or skeletal muscle toxicity. At the high dose of 9E13 vg/kg (approximately 3-fold higher than the efficacious dose of 3E13 vg/kg in this study), Compound A-related mild cardiomyocyte degeneration/necrosis was observed. These microscopic findings occurred without changes in biomarkers of cardiotoxicity and in the presence of continued functional (efficacy) improvement.

3. Single dose IV exploratory toxicity and biodistribution study (6-weeks) in cynomologus monkeys at 4E13 and 1.3E14 vg/kg Compound A.

Male and female cynomolgus monkeys, approximately 3-4 years old at initiation of dosing, were administered either a single dose of vehicle or Compound A by IV injection. The objective of this study was to investigate the potential toxicological effects as well as biodistribution in cynomolgus monkeys following a single IV dose of Compound A at 3E13 and 1 E14 vg/kg. Important to note that based on re-titer analysis of the lot completed after the study, the stock concentration was revised from 5.02E13 vg/mL to 6.7E13 vg/mL so that the intended doses of 3E13 and 1 E14 vg/kg were actually 4E13 vg/kg and 1.3E14 vg/kg. All groups were administered methylprednisolone once daily at 5 mg/kg by IM injection starting the day prior to and continuing through to the day prior to necropsy.

Table 10.

Animals were prescreened for anti-AAV9 neutralizing antibodies (nAb) prior to dosing and negative animals selected on study. The nAb evaluation used cell-based transduction inhibition assay. The nAb titers were reported as the lowest reciprocal serum dilution (5, 10, 20, 40) that had > 50% transduction inhibition. Samples with titers <5 were considered as nAb negative for AAV9. However, upon re-check post dosing, the high dose female (1 .3E14 vg/kg) was confirmed to have seroconverted with a high AAV9 nAb titer which accounts for the lack of target protein expression observed in this animal and impacted immune endpoint evaluation.

All animals survived to scheduled necropsy with no Compound A-related clinical observations. There were no Compound A-related macroscopic or microscopic findings.

There were Compound A-related increases in ALT, AST, GLDH, CK, cardiac troponin I, neutrophils, monocytes at > 4E13 vg/kg.

At 4E13 vg/kg, ALT was increased on Days 3 (female) and 8 (male, female) and returned to baseline by Day 15 (FIG. 11 A). At 1.3E14 vg/kg, ALT was increased on Day 15 (peak) and remained above baseline through Day 43 in the male only. AST was increased on Day 3 (female) at 4E13 vg/kg or Day 8 (male) at 1 .3E14 vg/kg and returned to baseline by Day 15. GLDH was also transiently increased on Days 8 (peak) and 15 in the male at 1 .3E14 vg/kg and on Days 3 (peak) and 8 in the female at 4E13 vg/kg. The ALT and GLDH increases are indicative of hepatocellular injury, however there was no microscopic correlate from histopathology evaluation performed on liver samples (necropsy Day 44 or 47). In the male only, at 1.3E14 vg/kg, CK was increased on Days 3 (peak) and 15, and returned to baseline by Day 29. Cardiac troponin I was elevated in the female animal only at 4E13 vg/kg on Day 43 consistent with a rising trend at study end. In the male only at 1 .3E14 vg/kg, neutrophils, monocytes and LUC were increased on Days 3, 8, and/or 43.

Evaluation of cytokines and complement factors revealed Compound A-related changes at the high dose. There were no Compound A-related changes in cytokines or complement activation products in animals dosed with 4E13 vg/kg Compound A. At 1.3E14 vg/kg, increased TNF (FIG. 11B) was observed in the male (7.6x baseline; Day 15) and female (4.8-9.4x baseline; peak on Day 15), as well as an increase in SC5b-9 (53x baseline; Day 1 at 6 HPD) in the female. The complement activation (SC5b-9) in the high dose female (1 .3E14 vg/kg) was likely initiated by AAV9 nAbs and not considered directly related to Compound A administration. The AAV9 nAbs along with complement activation likely potentiated the earlier onset and greater magnitude of TNF increase observed in the high dose female compared to the high dose male. The nAb response from pre-dose samples revealed that this animal seroconverted prior to dosing, thus indicating the animal was no longer immunologically naive and potentially impacting subsequent immune endpoint evaluations. A positive AAV9 capsid ELISpot response was noted in this high dose animal and was likely a recall I memory cell mediated response as a result of prior AAV9 exposure as evidenced by seroconversion prior to Compound A administration. There were smaller, uncertain, ELISpot signals noted in the males at both dose levels that were not considered related to Compound A administration.

The biodistribution (VGC, RNA, BAG3 protein) of Compound A at 4E13 vg/kg and 1 .3E14 vg/kg was evaluated with select tissues shown in Table 11 below (see also FIG. 6A- F). Heart expression was observed at >4E13 vg/kg with protein translation in all animals except the high dose female due to seroconversion. Evidence of expression was observed in the liver (protein in male only) and DRG neurons (no protein observed) without histopathology findings.

Table 11 . Compound A VGC, RNA, and BAG3 protein in select cyno tissues

In situ hybridization for BAG3 RNA was performed on heart sections from males and females from all groups. The positive signal, present as punctate red dots (FIG. 12), was present in the nucleus and cytoplasm of cardiomyocytes for all Compound A-dosed animals, except for the high dose female. The intensity and extent of positive ISH labelling in cardiomyocytes varied across heart samples and individual animals. In general, labelling was higher (number of red dots per cardiomyocytes and/or number of cardiomyocytes with red dots) with increasing dose in the males and higher in the male than the female at 4E13 vg/kg. In addition to semi-quantitative evaluation of percent positive cardiomyocytes by a pathologist, digital image analysis was performed to quantify the percent of positive cardiomyocytes (FIG. 6D). There were moderate variations in the positive labeling between the heart sections from a given animal within a group. Percent of BAG3 ISH positive cardiomyocytes in each section in the male and female administered 4E13 vg/kg ranged from 45 to 92% and 20 to 56%, respectively. In the male administered 1 .3E14 vg/kg, these values ranged from 79 to 95%.

In summary, 4E13 and 1.3E14 vg/kg Compound A, administered by the intended route of clinical administration, was tolerated in monkeys at 6-weeks post IV infusion and in the presence of a daily IS regimen. There were no Compound A-related histopathology findings. Clinical pathology results indicated Compound A-related increases in ALT, AST, CK, cardiac troponin I, neutrophils, monocytes at > 4E13 vg/kg. In addition, at the high dose (1.3E14 vg/kg), there were changes in immune endpoints (cytokine, complement, ELISpot) and one high dose animal that had seroconverted prior to dosing. Heart in situ hybridization for BAG3 RNA revealed a high percentage of BAG3-positive cardiomyocytes at 4E13 vg/kg ranging from 20 to 92% and at 1 .3E14 vg/kg ranging from 79% to 95% across heart sections in the high dose male.

Example 7: Tissue distribution / Tropism

BAG3 biodistribution was evaluated following IV administration of Compound A up to 1.3E14 vg/kg (approximately 3.3xthe efficacious dose demonstrated in cKO mice) in the cyno ETS (see cyno ETS Table 11 and FIG. 6A-F). Viral genome copies (VGC) were present and generally increased dose-dependently in the heart, liver, skeletal muscle, gonads, spinal cord, and DRG (FIG. 6A). While there was evidence of VGC in these tissues, there were no histopathology findings. AAV9 vector-mediated BAG3 expression (RNA) was detected primarily in the liver and heart (FIG. 6C), while expression was also noted in skeletal muscle, spinal cord, and DRG (gonads were not evaluated for RNA). ISH demonstrated significant and dose-dependent BAG3 expression across cyno cardiomyocytes (FIG. 6D). While total BAG3 (conserved) protein was noted in the cyno heart, liver, skeletal muscle, spinal cord, and DRG, the human specific peptide confirmed that human BAG3 protein was primarily localized to the heart at both dose levels evaluated. Marginal human BAG3 protein was detected at the high dose in the liver and no protein in spinal cord or DRG (FIG. 6E-6F).

Example 8: Pharmacokinetics-Pharmacodynamics Relationship and Prediction of Efficacious Human Dose.

The dose-translation plan for Compound A involved two steps: First the efficacious levels of target (BAG3) expression and heart coverage were determined using data from disease model mouse (BAG3-cKO) dose-response studies. Second, the dose necessary to achieve the mouse-based efficacious BAG3 expression was determined using data from a cynomolgus monkey study that tested Compound A at two different dose levels to evaluate BAG3 expression dose-response in monkey heart.

This translational plan assumed that the BAG3 expression (mRNA and proteinefficacy relationship in the BAG3 cKO mouse disease model is representative of the same relationship in BAG3 DCM patients. In addition, it was assumed that the dose-BAG3 expression relationship in humans can be based on the observed dose-BAG3 relationship in cynomolgus monkeys. These assumptions and the data from BAG3 cKO mouse and cynomolgus monkey studies were integrated into a quantitative systems pharmacology model for AAV gene therapy (developed and validated using available literature and inhouse data) to produce the efficacious dose and dose-dependent BAG3 expression predictions as described below. It should be noted that since the number of animals in the cynomolgus monkey ETS study used to project clinical dose was small (n = 2 at 4E13 and n = 1 at 1 .3E14 vg/kg) the dose-predictions may be updated when data based on future studies.

Nonclinical Pharmacology and Therapeutic Level of BAG3 Expression

The target therapeutic level of BAG3 expression in heart was determined based on results from a dose-response efficacy study in the BAG3 cKO mouse model of disease as described above. The echocardiography-based changes in cardiac structure and function post-treatment in individual animals observed in this study were correlated with the corresponding BAG3 mRNA and protein expression measured in heart tissue at necropsy to determine the BAG3 expression-efficacy relationship (FIG. 13A-13D).

Specifically, human BAG3 protein expression in BAG3 cKO mouse heart (quantified using LC/MS assay specific for the human BAG3 protein) which increased from 0 (control) to 70% (9E13 vg/kg) of wild-type mouse heart BAG3 expression with Compound A treatment was related to an increase in ejection fraction from 29% to 49%. The same improvement in ejection fraction also correlated with an increase in human BAG3 transgene heart tissue spatial coverage determined using in situ hybridization (human BAG3 mRNA; 52% cardiomyocytes hBAG3 positive at 9E13 vg/kg) and immunohistochemistry (BAG3 protein; 52% heart region hBAG3 positive at 9E13 vg/kg). All measures of protein and mRNA expression were also correlated with each other across dose groups.

Notably the relationship between human BAG3 mRNA/protein expression and improvement in cardiac function tended to saturation above approximately 20% wild-type mouse heart BAG3 protein expression and approximately 20% human BAG3 mRNA+ cardiomyocytes as determined by ISH (FIG. 13A-13B). Comparison of efficacy in individual mice binned by hBAG3 protein or ISH expression showed that mice that had >20% expression level by either measurement had a better ejection fraction change than mice that had <20% expression but were comparable to mice that had >40% expression (FIG. 13C- 13D). These results suggested that cardiac function improvement saturates around approximately 20% BAG3 expression level as measured by LC/MS-based bulk protein level and ISH-based cardiomyocyte coverage and therefore this level of expression was selected as the target therapeutic level of BAG3 expression.

Human Dosing

To determine the human dose necessary to achieve the target therapeutic levels of BAG3 expression data from the dose-response study of Compound A in cynomolgus monkeys together with a quantitative systems pharmacology (QSP) model developed was used and validated using available literature and in-house data for cross-species translation of AAV gene therapies.

In the cynomolgus monkey ETS, BAG3 biodistribution was evaluated following IV administration of Compound A at 4E13 and 1.3E14 vg/kg (doses based on final viral titer determined post-study - updated from original study design of 3E13 and 1 E14 vg/kg). The observed heart transduction of Compound A, measured as viral genome copy number per diploid genome equivalent (VCN/dge), increased dose-linearly in cynomolgus monkeys (FIG. 14A). The observed transduction was consistent with the model-predicted heart transduction for AAV9 as well as historical literature and in-house data for this capsid. This enabled use of this model to predict heart transduction for Compound A in humans. Note that the model predictions and available clinical data for AAV9 gene therapies indicated that heart transduction dose-dependence for Compound A can be expected to be very similar between non-human primates and humans.

The observed heart cardiomyocyte coverage of Compound A, measured using ISH for hBAG3 mRNA, also increased dose-dependently in cynomolgus monkeys (FIG. 14B). Heart tissue coverage exceeded the target level of >20% card io myocytes at both 4E13 and 1.3E14 vg/kg doses in monkeys and was anticipated to be translatable between monkeys and humans. Therefore any Compound A dose equal to or greater than 4E13 vg/kg was anticipated to be sufficient for achieving this therapeutic target level.

A Compound A-dose-dependent increase was also observed for hBAG3 protein level (measured using LC/MS) in the cynomolgus monkey hearts (FIG. 14C). The increase in protein level was greater than dose-linear (7x increase from 4E13 to 1 .3E14 vg/kg) although data was only available for one monkey at the high dose. This data was modeled using a power-law and included in the QSP model allowed us to project the Compound A dosedependent BAG3 protein expression in monkeys as shown in FIG. 14C.

To extend projections to humans, endogenous BAG3 protein expression was measured in untreated healthy cynomolgus monkey and healthy human heart samples. These measurements indicated an approximately 1.7-fold lower BAG3 protein level in human heart relative to monkey hearts (human:110 ng BAG3/mg protein vs cyno:190 ng BAG3/mg protein). A similar 1 ,7x lower protein expression efficiency for the human protein projection model was accordingly assumed. Based on these assumptions the model- predicted BAG3 protein dose-dependence in humans is shown in FIG. 14C. Using this predicted dose-dependence the clinical dose necessary to achieve the target level of BAG3 protein expression in human heart (22 ng BAG3/mg protein based on 20% of 110 ng BAG3/mg protein in normal healthy human heart) is projected to be about 1 E14 vg/kg of Compound A delivered intravenously. Note that if the monkey to human correction factor of 1 ,7x was not used and instead human protein expression was assumed to follow the cynomolgus monkey dose-dependence the projected clinical dose would be about 7E13 vg/kg of Compound A delivered intravenously.

A smaller effect than maximal efficacy could still be beneficial to patients, however. For example, a difference from placebo in ejection fraction of 5% would be both measureable and clinically meaningful. At 3E13 vg/kg, the ejection fraction change is estimated to be about 5 - 10%. Thus, an estimated dose of approximately 3E13 vg/kg could define the lower end of the clinical dose range.

In sum, about 3E13-1 E14 vg/kg of Compound A is projected to be the minimum efficacious dose range that is predicted to achieve the target levels BAG3 protein expression and cardiomyocyte coverage necessary for therapeutic benefit in patients. There are three key sources of uncertainty in this dose projection. In some embodiments, a pharmaceutically effective amount of the rAAV vector ranges from about 1 E10 to about 1 E17 vector genomes per kilogram (vg/kg) of subject body weight.

Example 9: In Vivo Efficacy Study of Compound A in A BAG3 Cardiac Heterozygous Mouse Model

Primary pharmacology of Compound A in vivo was interrogated in a mouse model of disease to evaluate the dose response associated efficacy. The mouse model of disease was generated by cardiac specific knockout of one or both BAG3 alleles resulting in partial (BAG3 cHET mice) or complete (BAG3 cKO mice) knockdown of BAG3 expression in the heart. These mice either have partial BAG3 expression (approximately 50%, BAG3 cHET) or completely lack BAG3 expression (BAG3 cKO) in cardiomyocytes, develop cardiac dysfunction and dilatation, develop cardiac fibrosis, and express biomarkers of heart failure and fibrosis in the heart (Fang et al., J. Clin. Invest. 127(8): 3189-200 (2017)). BAG3 cKO mice develop the disease phenotype at a faster rate when compared to the BAG3 cHET mice.

To determine whether restoration of BAG3 expression in the heart via AAV gene delivery results in improvement of cardiac structure and function or improvement in survival, a dose-response efficacy study in the BAG3 cHET mouse model of disease was pursued. BAG3 cHET mice were administered Compound A at 3E13 vg/kg, and 1 E14 vg/Kg. Cardiac structure and function and survival were assessed longitudinally. At necropsy AAV biodistribution and transgene expression (mRNA, protein) were assessed in the heart. HSPB8 stabilization (a surrogate of BAG3 expression) and biomarkers of heart failure (Nppa, Nppb, Myh6, Myh7) and apoptosis (Col1 a1 , Col1a2, Postn, Fn1 , Timpl) were also assessed in the heart.

Methods for Dose-response Efficacy and Survival Study

BAG3 cHET mice at age 27-28 weeks were administered Compound A intravenously at 3E13 vg/kg (n:14), and 1 E14 vg/Kg (n:14). The study also included a no-treatment control cohort of BAG3 cHET mice (n:14) and a no-treatment control cohort of BAG3 cWT mice (n:15). Cardiac structure and function were assessed using echocardiography at 25-26 weeks (baseline, 2-weeks prior to treatment) and at 30-31 weeks (3-weeks post treatment), at 35-36 weeks (8-weeks post treatment), and at 40-41 weeks (13-weeks post treatment). Survival was monitored approximately 14-weeks post-treatment.

At necropsy, heart tissue was collected for molecular biology analysis: AAV biodistribution, and transgene expression (mRNA, protein). Prior to treatment mice were randomized into groups based on echocardiography readouts (ejection fraction, left ventricular end diastolic volume), body weight and gender.

Echocardiography Assessment

For echocardiography imaging, mice were induced to anesthetic state using isoflurane (3%) or sevoflurane (5 to 6%) in an induction chamber, and then anesthetic status was maintained at approximately 2% isoflurane or approximately 4 to 4.5% sevoflurane during animal preparation and image acquisition. While anesthetized, an animal was transferred onto a water-circulating heated blanket to remove hair around the left lateral and ventral thoracic area using a chemical hair removal agent. After hair removal, each animal was transferred onto a heated (approximately 34 to 35° C) platform for echocardiography. Transthoracic parasternal long axis B-mode and parasternal short axis M-mode images of the heart were acquired using 18-38 MHz transducer (VisualSonics MS400®) in Vevo 2100® or Vevo 3100® ultrasound machine to assess left ventricular (LV) structure and function. Anesthesia level was adjusted to maintain heart rate between 450 to 550 bpm during image acquisition. After image acquisition, mice were transferred to a warm cage for recovery. Post image acquisition, images were analyzed using Vevo lab® analysis software, or automated analysis tool. The following parameters were collected from image analysis: LV ejection fraction, fractional shortening, LV end diastolic and systolic volume, LV end diastolic and systolic area, LV posterior and anterior wall.

Survival Monitoring Assessment

General animal well-being was monitored once daily. In addition, general signs and symptoms of heart failure such as abnormal breathing, poor body condition (< 2 out 5), poor activity/not moving around the cage etc. was monitored 1 to 2 times per week. Echocardiography was performed once in 3 to 5 weeks to assess cardiac function and structure. During monitoring for signs of heart failure, all animals were scored using an inhouse heart failure scoring chart to grade level of disease burden:

Table 12. Survival monitoring

Tissue Analysis

Frozen tissue samples were used for DNA isolation using phenol/chloroform DNA extraction methods. The DNA pellet was washed with ice cold 70% ethanol and resuspended in nuclease free water. DNA concentration was measured with Qiaxpert.

RNA isolation from frozen tissue was prepared by homogenization of approximately 20 mg of tissue sample in 1 mL Trizol with the presence of a 5 mm stainless steel bead using a Tissuelyzer II set at frequency 25 Hz for 5 minutes. Lysates were transferred to phasemaker tubes and 200 mL chloroform was added. Samples were vortexed for 15 seconds, incubated at room temperature for 5 minutes, and centrifuged at 14,000 g for 5 min at 4°C. The clear supernatant was transfer to a new tube and processed with the RNeasy RNA mini kit per manufacturer instructions. DNAse treatment was performed on column for 20 min before elution with nuclease free water. RNA concentration was measured using Nanodrop spectrophotometer

Protein isolated from frozen tissue was prepared by homogenization of approximately 20 mg of tissue sample in 100 pL RIPA buffer containing 1x protease inhibitor with the presence of a 5 mm stainless steel bead using a Tissuelyzer II set at frequency 25 Hz or 5 minutes. Lysates were incubated in wet ice for 30 minutes, and centrifuged at full speed for 30 minutes at 4°C. The clear supernatant was transferred to a new tube. Protein concentration was measured using BOA assay Kit.

Vector Genome Quantification (VGC)

VGC quantification was performed using ddPCR using the primer and probe sequences to amplify BAG3 transgene in all samples. The forward primer sequence was: GGCTGGCCCTTCTTCGT (SEQ ID NO: 16). The reverse primer sequence was GCCCTCAGAAGGCACTCT (SEQ ID NO: 17). The probe sequence was CCACAATAGCAGAACCAC (SEQ ID NO: 18).

Quantification of Transgene mRNA (BAG3) Expression

Isolated RNA samples were reverse transcribed to generate a cDNA library using the Superscript IV First-Stand Synthesis System following manufacturer’s protocol. 100 or 400 ng of total RNA was used in a 20 pL reaction. Reverse transcription was performed at 23°C for 10 minutes, 55°C for 10 minutes, then inactivate the reaction at 80°C for 10 minutes for 5 minutes. cDNA samples were stored at -20° C until RT-PCR analysis. cDNA was diluted using nuclease-free H2O by 1 :10 or 1 :40 to make the final 2.5 ng RNA amount in each ddPCR reaction. Quantification of BAG3 transgene mRNA expression was determined using ddPCR with the same BAG3 primers and probes as described in above section and housekeeping gene mTBP primer probes. PCR reaction mixture containing ddPCR Supermix (no dUTP) and above primer probes was prepared in an Airclean 600 PCR workstation and added to a ddPCR 96-well plate. Droplet generation, PCR programs, and data acquisition were performed as described in above section of VGC assessment. The mRNA expression was calculated by the ratio of FAM to VIC channel.

Assessment Total BAG3/HSPB8 Protein Expression

Protein expression of total BAG3 (human and mouse), human BAG3, and HSPB8 was detected using Wes/Jess from ProteinSimple with optimized conditions (protein amount and antibody dilution) following the manufacturer’s instruction. GAPDH was detected as the loading control. The chemiluminescent intensity of each detected protein was generated automatically by the instrument software. The protein expression of total BAG3, human BAG3, and HSPB8 was quantified by the ratio of each protein to GAPDH.

Quantification of Biomarkers of Heart Failure and Fibrosis

A total of 400 ng of RNA was used to prepare cDNA with the kit Superscript IV Vilo RT with ezDNase by following manufacturer instructions resulting in a final concentration of 20 ng/pL cDNA. For each RT reaction and no RT control reaction 10 pL of gDNA reaction mix was prepared by combining 1 pL of 10x ezDNase buffer, 1 pL of ezDNase enzyme, RNA (400 ng), and nuclease free water. Samples were incubated at 37°C for 2 min and returned to ice. The RT and No RT reactions were then prepared; for each RT reaction 4 pL of SSIV Vilo Master Mix was combined with 6 pL of nuclease free water, and for each no RT control reaction 4 pL of SSIV Vilo No RT Control Master Mix was combined with 6 pL of nuclease free water. 10 pL of master mix (RT or No RT respectively) was mixed with the 10 uL gRNA reaction to bring the reaction to a 20 pL final volume. RT was performed at 25°C for 10 min, 50°C for 10 min, and 85°C for 5 min. Samples were stored at -20 until RT-qPCR analysis. cDNA was prepared in a 96-well PCR plate. qPCR master mixes were then prepared with the cDNA template. cDNA was diluted using nuclease-free H₂O by 1 :10 or 1 :40 to make the final 2.5 ng RNA amount in each ddPCR reaction. For each sample the following was mixed: 55 pL of TaqMan Fast Advanced Master Mix (2x), 2.5 pL of 20 ng/uL cDNA, and 52.5 pL of nuclease free water. 100 pL of prepared PCR mix was then loaded into each reservoir on the array card. A total of 7 samples and one no reverse transcription (RT) control was added to each plate (8 reservoirs total/plate). The array card was centrifuged to ensure all the liquid was at the bottom on the tubes. The array card was removed from the centrifuge and sealed using the TaqMan Array Card Sealer. After sealing, the fill reservoir strip was cut from the card using scissors. The Array Card was the run on a ViiA 7 qPCR instrument using the standard cycling protocol (50°C for 2 minutes, 95°C for 20 seconds, [95°C for 1 second, 60°C for 20 seconds] x 40) for an Array Card and TaqMan Fast Advanced Master Mix. Results were analyzed using relative quantification to the housekeeping gene HPRT.

Table 13: Primers.

Results and Discussion

BAG3 cHET mice (mixed gender) were treated with Compound A at 3E13 vg/Kg and 1 E14 vg/Kg (age: 27-28 weeks). The study also included a no-treatment control cohort of cHET mice and a no-treatment control cohort of cWT mice.

Cardiac function (ejection fraction) and structure (left ventricular end-diastolic & end- systolic volume) were assessed longitudinally using echocardiography (FIG. 15A-15C) at an age of 25-26 weeks (baseline, 2-weeks prior to treatment), at an age of 30-31 weeks (3- weeks post treatment), at an age of 35-36 weeks (8-weeks post treatment), and at and age of 40-41 weeks (13-weeks post treatment). When measuring cardiac ejection fraction at all three timepoints post-treatment, no significant differences were detected when comparing treated mice (3E13 vg/Kg or 1 E14 vg/Kg) and the non-treated BAG3 cHET control cohort. A significant decrease was detected within all cHET groups when comparing values collected at baseline and at 8- or 13-weeks post treatment. When measuring LVEDV at all three timepoints post-treatment, no significant differences were detected when comparing treated mice (3E13 vg/Kg or 1 E14 vg/Kg) and the non-treated BAG3 cHET control cohort. A significant increase was detected in the non-treated control group when comparing values collected at baseline and at 8- or 13-weeks post treatment. When measuring LVESV at all three timepoints post-treatment, no significant differences were detected when comparing treated mice (3E13 vg/Kg or 1 E14 vg/Kg) and the non-treated BAG3 cHET control cohort. A significant increase was detected in the non-treated BAG3 cHET control group when comparing values collected at baseline and at 8- or 13-weeks post treatment.

Survival was assessed longitudinally up to 42 weeks (approximately 14-weeks post treatment) based on a pre-defined set of criteria described above. No significant differences in survival were detected when comparing treated mice (3E13 vg/Kg or 1 E14 vg/Kg) and the non-treated BAG3 cHET control cohort (FIG. 16A). No significant differences in body weight were detected in these mouse groups (FIG. 16B).

Viral genome biodistribution was examined in heart tissue collected at necropsy from BAG3 cHET mice treated with Compound A at 3E13 vg/kg and 1 E14 vg/kg (FIG. 17A). A dose response in biodistribution was detected in the heart: 3E13 vg/kg (0.131 ±0.066 vg/mTFRC), 1 E14 vg/kg (0.371±0.107 vg/mTFRC).

Transgene mRNA expression was examined in heart tissue collected at necropsy from mice treated with Compound A at 3E13 vg/kg and 1 E14 vg/kg (FIG. 17B). A dose response in expression was detected in the heart: 3E13 vg/kg (10.81±6.18 vg/mTBP), 1 E14 vg/kg (50.889±14.607 vg/mTBP).

BAG3 protein expression was examined in heart tissue collected at necropsy from mice treated with Compound A at 3E13 vg/kg and 1 E14 vg/kg (FIG. 17C). Protein expression was normalized to the levels of BAG3 expression detected in the hearts of a cohort of wild type mice. A dose-responsive increase in BAG3 protein levels was observed in BAG3 cHET mouse hearts after treatment with Compound A. At the high dose level (1 E14 vg/Kg), approximately 90% (0.912±0.196) of the WT level of BAG3 was detected and at the low dose (3E13 vg/Kg), approximately 60% (0.607±0.134) of WT BAG3 levels was observed, approximately 40% (0.392±0.096) of the WT level of BAG3 was detected in notreated cHET BAG3 mice.

HSPB8 protein expression was examined in heart tissue collected at necropsy from mice treated with Compound A at 3E13 vg/kg and 1 E14 vg/kg (FIG. 17D). Protein expression was normalized to the levels of BAG3 expression detected in the hearts of a cohort of wild type mice. A dose-responsive increase in HSPB8 protein levels was observed in BAG3 cHET mouse hearts after treatment with Compound A. At the high dose level (1 E14 vg/Kg), approximately 85% (0.855±0.155) of the WT level of BAG3 was detected and at the low dose (3E13 vg/Kg), approximately 63% (0.637±0.117) of WT BAG3 levels was observed, approximately 46% (0.460±0.071) of the WT level of BAG3 was detected in notreated cHET BAG3 mice.

Biomarkers of fibrosis (Col1a1 , Col1 a2, Fn1 , Postn, Timpl) and heart failure (Nppa, Nppb, Myh7, Myh6) were assessed (RNA expression) in the heart of treated and untreated mice. When measuring biomarkers of fibrosis, no significant differences were detected when comparing treated mice (3E13 vg/Kg or 1 E14 vg/Kg) and the non-treated BAG3 cHET control cohort (FIG. 18A-18E). When measuring biomarkers of heart failure, no significant differences were detected when comparing treated mice (3E13 vg/Kg or 1 E14 vg/Kg) and the non-treated BAG3 cHET control cohort (FIG. 18F-18H).

Conclusion

Treatment of BAG3 cHET mice at 27-28 weeks with Compound A did not result in improvement of cardiac structure, cardiac function, or survival when compared to nontreated control BAG3 cHET mice. A dose dependent increase of viral genomes in the heart correlated with a dose-dependent increase in transgene mRNA and protein expression and restoration of HSPB8 protein expression. No significant improvement was detected in biomarkers of heart failure or fibrosis.

Example 10: In vivo survival and efficacy study of Compound A in a BAG3 cardiac knockout mouse model

Primary pharmacology of Compound A in vivo was interrogated in wild type mice to evaluate the dose response associated efficacy. The mouse model of disease was generated by cardiac specific knockout of BAG3 expression (BAG3 cKO mice). These mice lack BAG3 expression in cardiomyocytes, develop cardiac dysfunction and dilatation, develop cardiac fibrosis, and express biomarkers of heart failure and fibrosis in the heart (Fang et al., J. Clin. Invest. 127(8): 3189-200 (2017)).

To determine whether restoration of BAG3 expression in the heart via AAV gene delivery results in improved survival, a dose-response efficacy study in the cKO mouse model of disease was pursued. BAG3 cKO mice were administered Compound A at 3E13 vg/kg, and 1 E14 vg/kg. Survival, cardiac structure and function were assessed longitudinally, using echocardiography. At necropsy AAV biodistribution and transgene expression (mRNA, protein) were assessed in the heart. Methods for Dose-Response Survival Study

BAG3 cKO mice at age 14-15 weeks were administered Compound A intravenously at 3E13 vg/kg (n:16), and 1 E14 vg/kg (n:15). The study also included a control cohort of cKO mice which was administered vehicle (n:13). Survival was monitored approximately 21 weeks post-treatment. Cardiac structure and function were assessed using echocardiography at baseline (1-week prior to treatment) and at 2-, 5-, 9- and 13-weeks post treatment. At necropsy, heart tissue was collected for molecular biology analysis: AAV biodistribution, and transgene expression (mRNA, protein). Prior to treatment mice were randomized into groups based on echocardiography readouts (ejection fraction, left ventricular end diastolic volume), body weight and gender.

General methods

The following methods were performed as described in Example 9: DNA isolation, RNA isolation, protein preparation, VGC quantification, quantification of BAG3 transgene RNA expression, quantification of total BAG3 protein expression, echocardiography assessment and survival monitoring assessment.

Results and Discussion

BAG3 cKO mice (mixed gender) were treated with either vehicle or Compound A at 3E13 vg/Kg and 1 E14 vg/Kg (age: 14-15 weeks). Survival (FIG. 19A) was assessed longitudinally (up to approximately! 6 weeks post treatment) based on a pre-defined set of criteria (see Example 9). It was determined that vehicle-treated mice (Group 1) had a significantly lower survival rate than mice treated with Compound A at either 3E13 vg/Kg (Group 2) or 1 E14 vg/Kg (Group 3). No significant differences in body weight were detected in these mouse groups (FIG. 19B).

Cardiac function (ejection fraction) and structure (left ventricular end-diastolic & end- systolic volume) were assessed longitudinally using echocardiography (FIG. 20A-20C) at baseline and at 2-, 5-, 9-, and 13-weeks post treatment. At week 13 post treatment, a significant improvement in ejection fraction was detected in mice treated with Compound A at 3E13 vg/kg (41 ,0±17.1 %) and 1 E14 vg/kg (51 ,2±16.6%) as compared to vehicle-treated BAG3 cKO mice (27.0±15.8%). At week 13, a significant improvement in left ventricular end diastolic volume and left ventricular end systolic volume was also detected in mice treated with Compound A at 1 E14 vg/kg (LVEDV: 54.7±23.7 uL, LVESV: 29.8±26.5 uL) as compared to vehicle-treated BAG3 cKO mice (LVEDV: 75.6±25.1 uL, LVESV: 57.6±28.3 uL).

Viral genome biodistribution was examined in heart tissue collected at necropsy from mice treated with Compound A at 3E13 vg/kg and 1 E14 vg/kg (FIG. 21 A). A dose response in biodistribution was detected in the heart: 3E13 vg/kg (0.080±0.039 vg/mTFRC), 1 E14 vg/kg (0.32±0.13 vg/mTFRC).

Transgene mRNA expression was examined in heart tissue collected at necropsy from mice treated with Compound A at 3E13 vg/kg and 1 E14 vg/kg (FIG. 21 B). A dose response in expression was detected in the heart: 3E13 vg/kg (8.21 ±5.59 vg/mTFRC), 1 E14 vg/kg (43.91 ±15.99 vg/mTFRC).

BAG3 protein expression was also examined in heart tissue collected at necropsy from mice treated with Compound A at 3E13 vg/kg and 1 E14 vg/kg (FIG. 21 C). Protein expression was normalized to the levels of BAG3 expression detected in the hearts of a cohort of wild type mice. A dose-responsive increase in BAG3 protein levels was observed in cKO mouse hearts after treatment with Compound A. At the high dose level (1 E14 vg/Kg), approximately 50% (0.49±0.16) of the WT level of BAG3 was detected and at the low dose (3E13 vg/Kg), approximately 8% (0.08±0.04) of WT BAG3 levels was observed.

Conclusion

Treatment of BAG3 cKO mice with Compound A resulted in improvement in survival and a dose-dependent improvement of both cardiac structure and function that correlated with expression of human BAG3 protein in the heart.

Example 11 : Clinical Study

BAG3 is implicated in diverse cellular function including but not limited to excitation contraction coupling, maintenance of sarcomere integrity and regulation of autophagy. Comprehensive assessments of the manifestations of disease caused by BAG3 mutations in the general population are complicated by the rarity of the disease and the single center nature of most case reports of BAG3 associated disease. Precise characterization BAG3 DCM with imaging and circulating biomarkers at presentation as well as its clinical progression remains to be elucidated. The development of novel transformational therapies for BAG3 DCM requires clear understanding of circulating, imaging, and clinical biomarkers of disease status at baseline and overtime. As such, a longitudinal natural history study in patients diagnosed with dilated cardiomyopathy due to likely pathogenic or pathologic BAG3 mutations is being conducted to clarify these measures.

Eligible subjects from this longitudinal study may be enrolled in a 2-Part, Phase 1 b, Interventional, Open-Label, Dose-Ascending Study, Followed by a Phase 2, Placebo- Controlled, Double Blinded Study to Investigate the Safety, Tolerability, and Efficacy of a Single Dose of ALXN2350 in Participants with BAG3 Mutation Associated Dilated Cardiomyopathy.

This prospective, open-label, dose-evaluation Phase 1 b followed by blinded, placebo-controlled Phase 2 study is a first-in-human (FIH)/first-in-patient (FIP) study with adeno-associated virus, serotype 9 (AAV9)-based gene therapy Compound A. This study aims to evaluate safety, tolerability, and efficacy following a single dose of Compound A in participants with dilated cardiomyopathy attributable to likely pathogenic or pathogenic BAG3 mutations. Other objectives include exploratory assessments relevant to long-term safety, dose selection, quality of life, and functional capacity to support future clinical development.

A two-part design with sequential conduct of Phase 1 b and Phase 2, with each part conducted in distinct/separate cohorts of participants, is proposed. The Phase 1 b portion of the study aims to explore safety, tolerability and immunogenicity across the dose range from the starting projected minimally active dose of approximately 3E13 vg/kg to the maximum feasible dose of approximately 1 E14 vg/kg. To allow for a blinded assessment of efficacy, the Phase 2 portion of the study will compare the proposed clinical dose level discovered in the Phase 1 b to a matching placebo in a randomized double-blinded study design. After an initial 12 months of efficacy and safety follow up, all eligible participants in the Phase 2 portion that received placebo will be given the opportunity to cross over to intervention with Compound A to allow for expanded investigation of safety and efficacy (employing a delayed start analysis to further evaluate the impact of therapy on disease progression). The doubleblinded nature of the study will remain in place until all participants have completed at least 12-months of follow-up and an independent data monitoring committee has determined the suitability of open label extension.

The Phase 1 b/2 study will be followed by a phase 3 double blinded randomized placebo-controlled study with the primary efficacy endpoint of improvements in functional capacity and symptoms. In this pivotal phase 3 registrational study, an interim analysis will be conducted to examine the effect of the investigational medicinal product on positive cardiac remodeling. At this interim analysis, both futility as well as overwhelming success criteria will be leveraged. If the level of positive (restorative) remodeling achieves prespecified levels of success compared to placebo, this data will be leveraged for an early accelerated approval strategy.

Table 14. First in patient clinical plan

Table 15. Clinical biomarker plan

Selection of subjects Subjects included in the protocol must meet specific inclusion criteria that include but is not limited to the following: Males/Females; Known BAG-3 mutation (likely pathologic/pathologic); Age >18 years; LVEF <50%; Stage B (NYHA l/IV)

Subjects will be excluded if they have: CKD > I lib; Non BAG3 related Heart Disease; Transaminase >2 x ULN; T. Bili > 1 ,5x ULN.

Claims

WHAT IS CLAIMED IS:

1 . A nucleic acid molecule comprising a nucleotide sequence encoding a Bcl2- associated athanogene 3 (BAG3) polypeptide, or variant thereof.

2. The nucleic acid molecule of claim 1 , wherein the nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is a codon-optimized nucleotide sequence.

3. The nucleic acid molecule of either claim 1 or claim 2, wherein the nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is at least about 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4 or 19- 24.

4. The nucleic acid molecule of any one of claims 1-3, wherein the nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is at least about 80% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

5. The nucleic acid molecule of any one of claims 1-4, wherein the nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is at least about 85% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

6. The nucleic acid molecule of any one of claims 1-5, wherein the nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is at least about 90% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

7. The nucleic acid molecule of any one of claims 1-6, wherein the nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is at least about 95% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

8. The nucleic acid molecule of any one of claims 1-7, wherein the nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is at least about 98% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

9. The nucleic acid molecule of any one of claims 1-8, wherein the nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is at least about 99% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

10. The nucleic acid molecule of any one of claims 1-9, wherein the nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is the nucleotide sequence of SEQ ID NO: 4 or 19-24.

11. A recombinant adeno-associated virus (rAAV) vector comprising the nucleic acid molecule of any one of claims 1-10.

12. The rAAV vector of claim 11 , wherein the rAAV vector comprises serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), or serotype 12 (AAV12) capsid protein.

13. The rAAV vector of either claim 11 or claim 12, wherein the rAAV vector comprises AAV9 capsid protein.

14. The rAAV vector of claim 13, wherein the AAV9 capsid protein is a VP1 protein comprising the amino acid sequence of SEQ ID NO: 1 or a functional subsequence, modification, or variant thereof, a VP2 protein comprising the amino acid sequence of SEQ ID NO: 2 or a functional subsequence, modification, or variant thereof, or a VP3 protein comprising the amino acid sequence of SEQ ID NO: 3 or a functional subsequence, modification, or variant thereof.

15. The rAAV vector of either claim 13 or claim 14, wherein the AAV9 capsid protein comprises the VP1 protein comprising the amino acid sequence of SEQ ID NO: 1 or a functional subsequence, modification, or variant thereof, the VP2 protein comprising the amino acid sequence of SEQ ID NO: 2 or a functional subsequence, modification, or variant thereof, and the VP3 protein comprising the amino acid sequence of SEQ ID NO: 3 or a functional subsequence, modification, or variant thereof.

16. The rAAV vector of any one of claims 11-15, wherein the rAAV vector further comprises at least one cardiac promoter operably linked to the nucleic acid molecule comprising the nucleotide sequence encoding a BAG3 polypeptide, or variant thereof.

17. The rAAV vector of claim 16, wherein the at least one cardiac promoter is chicken troponin T (cTNT), CAG, MHCK7, CK7, endogenous BAG promoter, desmin (Des), alphamyosin heavy chain (a-MHC), myosin light chain 2 (MLC-2), cardiac troponin C (TNNC1 or cTnC), human cardiac troponin T (TNNT2) promoter, or a functional subsequence, modification, or variant respectively thereof.

18. The rAAV vector of either claim 16 or claim 17, wherein the at least one cardiac promoter is cTNT promoter or a functional subsequence, modification, or variant thereof.

19. The rAAV vector of either claim 17 or claim 18, wherein the cTNT promoter comprises the nucleotide sequence of SEQ ID NO: 5, or a functional subsequence, modification, or variant thereof.

20. The rAAV vector of either claim 17 or claim 18, wherein the cTNT promoter comprises the nucleotide sequence of SEQ ID NO: 5.

21 . The rAAV vector of any one of claims 11-20, wherein the rAAV vector further comprises at least one intron.

22. The rAAV vector of claim 21 , wherein the intron comprises the nucleotide sequence of SEQ ID NO: 6, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31 , or a functional subsequence, modification, or variant thereof.

23. The rAAV vector of either claim 21 or claim 22, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 6.

24. The rAAV vector of either claim 21 or claim 22, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 29.

25. The rAAV vector of either claim 21 or claim 22, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 30.

26. The rAAV vector of either claim 21 or claim 22, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 31 .

27. The rAAV vector of any one of claims 11-26, wherein the rAAV vector further comprises at least one 5’ inverted terminal repeat (ITR) sequence.

28. The rAAV vector of claim 27, wherein the at least one 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 8, or a functional subsequence, modification, or variant thereof.

29. The rAAV vector of either claim 27 or claim 28, wherein the at least one 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 8.

30. The rAAV vector of any one of claims 11-29, wherein the rAAV vector further comprises at least one 3’ ITR sequence.

31 . The rAAV vector of claim 30, wherein the at least one 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 9, or a functional subsequence, modification, or variant thereof.

32. The rAAV vector of either claim 30 or claim 31 , wherein the at least one 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 9.

33. The rAAV vector of any one of claims 11-32, wherein the rAAV vector further comprises at least one transcription terminator sequence.

34. The rAAV vector of claim 33, wherein the at least one transcription terminator sequence is an SV40 polyA sequence, a bovine growth hormone (BGH) polyA sequence, a rabbit b-globin (rPg) polyA sequence, or a functional subsequence, modification, or variant thereof.

35. The rAAV vector of either claim 33 or claim 34, wherein the at least one transcription terminator sequence is an SV40 polyA sequence.

36. The rAAV vector of any one of claims 33-35, wherein the at least one transcription terminator sequence comprises the nucleotide sequence of SEQ ID NO: 7, or a functional subsequence, modification, or variant thereof.

37. The rAAV vector of any one of claims 33-36, wherein the at least transcription terminator sequence comprises the nucleotide sequence of SEQ ID NO: 7.

38. The rAAV vector of any one of claims 11-37, wherein the rAAV vector further comprises at least one stuffer or filler sequence, preferably wherein the at least one stuffer of filler sequence increases the entire length of the nucleic acid molecule of the rAAV vector, inclusive of the ITR sequences, to approximately 4.2 to 4.7 kilobases.

39. The rAAV vector of claim 38, wherein the at least one stuffer or filler sequence comprises the nucleotide sequence of SEQ ID NO: 11 , or a functional subsequence, modification, or variant thereof.

40. The rAAV vector of either claim 38 or claim 39, wherein the at least one stuffer or filler sequence comprises the nucleotide sequence of SEQ ID NO: 11 .

41 . An rAAV vector comprising, in 5’ to 3’ order:

(a) at least one 5’ ITR sequence;

(b) at least one cardiac promoter;

(c) at least one intron;

(e) at least one transcription terminator sequence;

(f) at least one stuffer or filler sequence; and

(g) at least one 3’ ITR sequence.

42. The rAAV vector of claim 41 , wherein the rAAV vector comprises serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), or serotype 12 (AAV12) capsid protein.

43. The rAAV vector of either claim 41 or claim 42, wherein the rAAV vector comprises AAV9 capsid protein.

44. The rAAV vector of claim 43, wherein the AAV9 capsid protein is a VP1 protein comprising the amino acid sequence of SEQ ID NO: 1 or a functional subsequence, modification, or variant thereof, a VP2 protein comprising the amino acid sequence of SEQ ID NO: 2 or a functional subsequence, modification, or variant thereof, or a VP3 protein comprising the amino acid sequence of SEQ ID NO: 3 or a functional subsequence, modification, or variant thereof.

45. The rAAV vector of either claim 43 or claim 44, wherein the AAV9 capsid protein comprises the VP1 protein comprising the amino acid sequence of SEQ ID NO: 1 or a functional subsequence, modification, or variant thereof, the VP2 protein comprising the amino acid sequence of SEQ ID NO: 2 or a functional subsequence, modification, or variant thereof, and the VP3 protein comprising the amino acid sequence of SEQ ID NO: 3 or a functional subsequence, modification, or variant thereof.

46. An rAAV vector plasmid comprising, in 5’ to 3’ order:

(a) at least one left spacer sequence;

(b) the at least one 5’ ITR sequence;

(c) the at least one cardiac promoter;

(d) the at least one intron;

(f) the at least one transcription terminator sequence;

(g) the at least one stuffer or filler sequence;

(h) the at least one 3’ ITR sequence; and

(i) at least one right spacer sequence.

47. The rAAV vector of any one of claims 41 -45 or rAAV vector plasmid of claim 46, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is a codon-optimized nucleotide sequence.

48. The rAAV vector of any one of claims 41-45 or 47 or rAAV vector plasmid of any one of claims 46 or 47, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is at least about 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

49. The rAAV vector of any one of claims 41 -45, 47, or 48 or rAAV vector plasmid of any one of claims 46-48, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is at least about 80% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

50. The rAAV vector of any one of claims 41 -45 or 47-49 or rAAV vector plasmid of any one of claims 46-49, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is at least about 85% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

51 . The rAAV vector of any one of claims 41 -45 or 47-50 or rAAV vector plasmid of any one of claims 46-50, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is at least about 90% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

52. The rAAV vector of any one of claims 41 -45 or 47-51 or rAAV vector plasmid of any one of claims 46-51 , wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is at least about 95% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

53. The rAAV vector of any one of claims 41 -45 or 47-52 or rAAV vector plasmid of any one of claims 46-52, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is at least about 98% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

54. The rAAV vector of any one of claims 41 -45 or 47-53 or rAAV vector plasmid of any one of claims 46-53, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is at least about 99% identical to the nucleotide sequence of SEQ ID NO: 4 or 19-24.

55. The rAAV vector of any one of claims 41 -45 or 47-54 or rAAV vector plasmid of any one of claims 46-54, wherein the at least one nucleotide sequence encoding the BAG3 polypeptide, or variant thereof, is the nucleotide sequence of SEQ ID NO: 4 or 19-24.

56. The rAAV vector of any one of claims 41 -45 or 47-55 or rAAV vector plasmid of any one of claims 46-55, wherein the at least one 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 8, or a functional subsequence, modification, or variant thereof.

57. The rAAV vector of any one of claims 41 -45 or 47-56 or rAAV vector plasmid of any one of claims 46-56, wherein the at least one 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 8.

58. The rAAV vector of any one of claims 41 -45 or 47-57 or rAAV vector plasmid of any one of claims 46-57, wherein the at least one cardiac promoter is chicken troponin T (cTNT), CAG, MHCK7, CK7, endogenous BAG promoter, desmin (Des), alpha-myosin heavy chain (a-MHC), myosin light chain 2 (MLC-2), cardiac troponin C (TNNC1 or cTnC), human cardiac troponin T (TNNT2) promoter, or a functional subsequence, modification, or variant thereof.

59. The rAAV vector or rAAV vector plasmid of claim 58, wherein the at least one cardiac promoter is cTNT promoter or a functional subsequence, modification, or variant thereof.

60. The rAAV vector or rAAV vector plasmid of either claim 58 or claim 59, wherein the cTNT promoter comprises the nucleotide sequence of SEQ ID NO: 5, or a functional subsequence, modification, or variant thereof.

61 . The rAAV vector or rAAV vector plasmid of any one of claims 58-60, wherein the cTNT promoter comprises the nucleotide sequence of SEQ ID NO: 5.

62. The rAAV vector of any one of claims 41 -45 or 47-61 or rAAV vector plasmid of any one of claims 46-61 , wherein the at least one intron comprises the nucleotide sequence of SEQ ID NO: 6, or a functional subsequence, modification, or variant thereof.

63. The rAAV vector of any one of claims 41 -45 or 47-62 or rAAV vector plasmid of any one of claims 46-62, wherein the at least one intron comprises the nucleotide sequence of SEQ ID NO: 6.

64. The rAAV vector of any one of claims 41 -45 or 47-63 or rAAV vector plasmid of any one of claims 46-63, wherein the intron comprises the nucleotide sequence of SEQ ID NO:

29.

65. The rAAV vector of any one of claims 41 -45 or 47-64 or rAAV vector plasmid of any one of claims 46-64, wherein the intron comprises the nucleotide sequence of SEQ ID NO:

30.

66. The rAAV vector of any one of claims 41 -45 or 47-65 or rAAV vector plasmid of any one of claims 46-65, wherein the intron comprises the nucleotide sequence of SEQ ID NO:

31.

67. The rAAV vector of any one of claims 41 -45 or 47-66 or rAAV vector plasmid of any one of claims 46-66, wherein the at least one transcription terminator sequence is an SV40 polyA sequence, a bovine growth hormone (BGH) polyA sequence, a rabbit b-globin (rPg) polyA sequence, or a functional subsequence, modification, or variant thereof.

68. The rAAV vector of any one of claims 41 -45 or 47-67 or rAAV vector plasmid of any one of claims 46-67, wherein the at least one transcription terminator sequence is an SV40 polyA sequence.

69. The rAAV vector of any one of claims 41 -45 or 47-68 or rAAV vector plasmid of any one of claims 46-68, wherein the at least one transcription terminator sequence comprises the nucleotide sequence of SEQ ID NO: 7, or a functional subsequence, modification, or variant thereof.

70. The rAAV vector of any one of claims 41 -45 or 47-69 or rAAV vector plasmid of any one of claims 46-69, wherein the at least transcription terminator sequence comprises the nucleotide sequence of SEQ ID NO: 7.

71 . The rAAV vector of any one of claims 41-45 or 47-70 or rAAV vector plasmid of any one of claims 46-70, wherein at least one stuffer or filler sequence increases the entire length of the nucleic acid molecule of the rAAV vector or rAAV vector plasmid, inclusive of the ITR sequences and excluding the spacer sequences, to approximately 4.2 to 4.7 kilobases.

72. The rAAV vector of any one of claims 41-45 or 47-71 or rAAV vector plasmid of any one of claims 46-71 , wherein the at least one stuffer or filler sequence comprises the nucleotide sequence of SEQ ID NO: 11 , or a functional subsequence, modification, or variant thereof.

73. The rAAV vector of any one of claims 41 -45 or 47-72 or rAAV vector plasmid of any one of claims 46-72, wherein the at least one stuffer or filler sequence comprises the nucleotide sequence of SEQ ID NO: 11 .

74. The rAAV vector of any one of claims 41 -45 or 47-73 or rAAV vector plasmid of any one of claims 46-73, wherein the at least one 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 9, or a functional subsequence, modification, or variant thereof.

75. The rAAV vector of any one of claims 41 -45 or 47-74 or rAAV vector plasmid of any one of claims 46-74, wherein the at least one 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 9. Ill

76. The rAAV vector plasmid of any one of claims 46-75, wherein the at least one left spacer sequence comprises the nucleotide sequence of SEQ ID NO: 12, or a functional subsequence, modification, or variant thereof.

77. The rAAV vector plasmid of any one of claims 46-76, wherein the at least one left spacer sequence comprises the nucleotide sequence of SEQ ID NO: 12.

78. The rAAV vector plasmid of any one of claims 46-77, wherein the at least one right spacer sequence comprises the nucleotide sequence of SEQ ID NO: 13, or a functional subsequence, modification, or variant thereof.

79. The rAAV vector plasmid of any one of claims 46-78, wherein the at least one right spacer sequence comprises the nucleotide sequence of SEQ ID NO: 13.

80. The rAAV vector plasmid of any one of claims 46-79, wherein the rAAV vector plasmid further comprises at least one left spacer and/or at least one right spacer, preferably wherein the at least one left spacer and/or at least one right spacer increases the entire length of the rAAV vector plasmid to approximately 4.7 to 12 kilobases, preferably to approximately 9 to 10 kilobases.

81 . The rAAV vector of any one of claims 11 -45 or 47-75 or rAAV vector plasmid of any one of claims 46-80, wherein the rAAV vector or rAAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 14, or a functional subsequence, modification, or variant thereof.

82. The rAAV vector of any one of claims 11 -45, 47-75, or 81 or rAAV vector plasmid of any one of claims 46-81 , wherein the rAAV vector or rAAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 14.

83. The rAAV vector of any one of claims 11 -45, 47-75, 81 , or 82 or rAAV vector plasmid of any one of claims 46-82, wherein the rAAV vector or rAAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 15, or a functional subsequence, modification, or variant thereof.

84. The rAAV vector of any one of claims 11-45, 47-75, or 81-83 or rAAV vector plasmid of any one of claims 46-83, wherein the rAAV vector or rAAV vector plasmid comprises the nucleotide sequence of SEQ ID NO: 15.

85. The rAAV vector of any one of claims 11-45, 47-75, or 81-84, wherein the entire length of the nucleic acid molecule of the rAAV vector is equal to or less than approximately 5 kilobases in length or is equal to or less than 4.7 kilobases in length.

86. The rAAV vector plasmid of any one of claims 46-84, wherein the entire length of the rAAV vector plasmid is equal to or less than approximately 12 kilobases in length or is equal to or less than 4.7 kilobases in length.

87. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1-10, or the rAAV vector of any one of claims 11-45, 47-75, or 81-85, and at least one pharmaceutically acceptable salt.

88. The pharmaceutical composition of claim 87, wherein the at least one pharmaceutically acceptable salt is present in an amount ranging from about 1 mM to about 450 mM.

89. The pharmaceutical composition of either claim 87 or claim 88, wherein the at least one pharmaceutically acceptable salt is present in an amount ranging from about 2 mM to about 350 mM.

90. The pharmaceutical composition of any one of claims 87-89, wherein the at least one pharmaceutically acceptable salt is sodium chloride, magnesium chloride, potassium chloride, calcium chloride, or calcium phosphate.

91 . The pharmaceutical composition of claim 90, wherein the at least one pharmaceutically acceptable salt comprises sodium chloride and magnesium chloride.

92. The pharmaceutical composition of claim 90, wherein the at least one pharmaceutically acceptable salt comprises sodium chloride and potassium chloride.

93. The pharmaceutical composition of any one of claims 87-92, further comprising at least one buffer.

94. The pharmaceutical composition of claim 93, wherein the buffer is citrate, histidine, acetate, phosphate, tris hydrochloride, or tromethamine.

95. The pharmaceutical composition of claim 94, wherein the buffer is phosphate.

96. The pharmaceutical composition of claim 94, wherein the buffer is tris hydrochloride and tromethamine.

97. The pharmaceutical composition of any one of claims 93-96, wherein the at least one buffer is present in an amount ranging from about 10 mM to about 40 mM.

98. The pharmaceutical composition of claim 97, wherein the at least one buffer is present in an amount of about 20 mM.

99. The pharmaceutical composition of any one of claims 87-98, further comprising at least one cryoprotectant.

100. The pharmaceutical composition of claim 99, wherein the at least one cryoprotectant is a sugar or a sugar alcohol.

101 . The pharmaceutical composition of claim 99, wherein the at least one cryoprotectant is trehalose, sucrose, sorbitol, or mannitol.

102. The pharmaceutical composition of claim 101 , wherein the at least one cryoprotectant is sucrose.

103. The pharmaceutical composition of claim 101 , wherein the at least one cryoprotectant is sorbitol.

104. The pharmaceutical composition of any one of claims 99-103, wherein the at least one cryoprotectant is present in an amount of up to about 20%.

105. The pharmaceutical composition of claim 104, wherein the at least one cryoprotectant is present in an amount ranging from about 3% to about 15%.

106. The pharmaceutical composition of claim 105, wherein the at least one cryoprotectant is present in an amount of about 4% or about 5%.

107. The pharmaceutical composition of any one of claims 87-106, further comprising at least one surfactant.

108. The pharmaceutical composition of claim 107, wherein the at least one surfactant is a polaxamer or a polysorbate.

109. The pharmaceutical composition of claim 108, wherein the at least one surfactant is polaxamer 188, polysorbate 20, or polysorbate 80.

110. The pharmaceutical composition of any one of claims 107-109, wherein the at least one surfactant is present in an amount ranging from about 0.0001% to about 1 %.

111. The pharmaceutical composition of claim 110, wherein the at least one surfactant is present in an amount of about 0.02%.

112. The pharmaceutical composition of claim 110, wherein the at least one surfactant is present in an amount of about 0.002%.

113. The pharmaceutical composition of any one of claims 87-112, wherein the pharmaceutical composition has a pH ranging from about 6 to about 8.

114. The pharmaceutical composition of claim 113, wherein the pharmaceutical composition has a pH ranging from about 7 to about 8.

115. The pharmaceutical composition of claim 114, wherein the pharmaceutical composition has a pH of about 7.6.

116. The pharmaceutical composition of claim 114, wherein the pharmaceutical composition has a pH of about 7.4.

117. A method for treating a cardiac-related disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of:

(a) the nucleic acid molecule of any one of claims 1-10; or

(b) the rAAV vector of any one of claims 11 -45, 47-75, or 81-85; or

(c) the pharmaceutical composition of any one of claims 87-116.

118. The method of claim 117, wherein the cardiac-related disease or disorder is associated with a deficiency or dysfunction of BAG3.

119. The method of either claim 117 or claim 118, wherein the subject has a BAG3 mutation.

120. The method of any one of claims 117-119, wherein the cardiac-related disease or disorder is BAG3-related dilated cardiomyopathy (DCM).

121. The method of any one of claims 117-119, wherein the cardiac-related disease or disorder is BAG3-related heart failure.

122. The method of claim 117, wherein the cardiac-related disease or disorder is not associated with a deficiency or dysfunction of BAG3.

123. The method of either claim 117 or claim 122, wherein the subject does not have a BAG mutation.

124. The method of any one of claims 117, 122, or 123, wherein the cardiac-related disease or disorder is heart failure unrelated to BAG3 expression.

125. A method for reducing the frequency or severity of at least one symptom associated with a cardiac-related disease or disorder in a subject, the method comprising administering to the subject:

(a) the nucleic acid molecule of any one of claims 1-10; or

(b) the rAAV vector of any one of claims 11 -45, 47-75, or 81-85; or

(c) the pharmaceutical composition of any one of claims 87-116; in an amount effective to reduce the frequency or severity of the at least one symptom.

126. The method of claim 125, wherein the cardiac-related disease or disorder is associated with a deficiency or dysfunction of BAG3.

127. The method of either claim 125 or claim 126, wherein the subject has a BAG3 mutation.

128. The method of any one of claims 125-127, wherein the cardiac-related disease or disorder is BAG3-related dilated cardiomyopathy (DCM).

129. The method of any one of claims 125-127, wherein the cardiac-related disease or disorder is BAG3-related heart failure.

130. The method of claim 125, wherein the cardiac-related disease or disorder is not associated with a deficiency or dysfunction of BAG3.

131. The method of either claim 125 or claim 130, wherein the subject does not have a BAG mutation.

132. The method of any one of claims 125, 130, or 131 , wherein the cardiac-related disease or disorder is heart failure unrelated to BAG3 expression.

133. The method of any one of claims 125-128, wherein the at least one symptom is characteristic of BAG3-related DCM.

134. The method of any one of claims 125-127 or 129, wherein the at least one symptom is characteristic of BAG3-related heart failure.

135. The method of any one of claims 125 or 130-132, wherein the at least one symptom is characteristic of heart failure unrelated to BAG3 expression.

136. The method of any one of claims 117-135, wherein the effective amount of the rAAV vector ranges from about 1 E10 to about 1 E17 vector genomes per kilogram (vg/kg) of subject body weight.

137. The method of claim 136, wherein the effective amount of the rAAV vector ranges from about 3E13 to about 1 E14 vector genomes per kilogram (vg/kg) of subject body weight.

138. The method of either claim 136 or claim 137, wherein the effective amount of the rAAV vector is about 3E13 vector genomes per kilogram (vg/kg) of subject body weight.

139. The method of either claim 136 or claim 137, wherein the effective amount of the rAAV vector is about 7E13 vector genomes per kilogram (vg/kg) of subject body weight.

140. The method of either claim 136 or claim 137, wherein the effective amount of the rAAV vector is 1 E14 vector genomes per kilogram (vg/kg) of subject body weight.

141 . Use of the nucleic acid molecule of any one of claims 1-10 in the manufacture of a medicament for treating a cardiac-related disease or disorder in a subject.

142. Use of the rAAV vector of any one of claims 11-45, 47-75, or 81-85 in the manufacture of a medicament for treating a cardiac-related disease or disorder in a subject.

143. Use of the pharmaceutical composition of any one of claims 87-116 in the manufacture of a medicament for treating a cardiac-related disease or disorder in a subject.

144. A plasmid comprising the nucleic acid molecule of any one of claims 1-10.

145. A plasmid comprising the rAAV vector sequence of any one of claims 11-45, 47-75, or 81-85.

146. A host cell for rAAV vector production comprising the plasmid of claim 144.

147. A host cell for rAAV vector production comprising the plasmid of claim 145.

148. A host cell for rAAV vector production comprising the rAAV vector plasmid of any one of claims 46-84.

149. The host cell of any one of claims 146-148, wherein the host cell is an HEK293 cell or derivative thereof.

150. The host cell of any one of claims 146-149, wherein the host cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding an AAV Rep protein.

151 . The host cell of any one of claims 146-149, wherein the host cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding an AAV9 capsid protein.

152. The host cell of any one of claims 146-149, wherein the host cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding a viral helper factor.

153. A method of making an rAAV vector comprising:

(a) incubating the host cell of any one of claims 146-152 under conditions sufficient to allow the production of rAAV vectors; and

(b) purifying the rAAV vectors produced thereby.

154. An rAAV vector produced by the method of claim 152.