Novel human heart-derived adeno-associated viruses are cardiac-specific gene carriers for effective in vivo gene delivery to mammalian hearts and human iPSC-derived cardiomyocytes.

Introduction:
Adeno-associated viral (AAV) vectors used in current clinical trials for cardiac gene therapy (GT) lack myocardial specificity. AAV9, being investigated for human cardiomyopathy GT, exerts high hepatic toxicity which resulted in fatal liver failure and death in several patients. In this context, AAVs with high cardiac specificity and the ability to transduce the human heart would represent ideal vectors for human cardiac GT.

Hypothesis:
Thus, we hypothesized that the human heart could be the best source for identifying novel AAVs capable to transduce human myocardium. These may have appropriate cardiac specificity allowing safe and effective gene transfer in mammalian hearts.

Approach and Results:
We identified AAV9 and AAV6 in heart failure patient myocardial biopsies as serotypes known to effectively transduce myocardium in vivo and in vitro, respectively. Additionally, a novel AAV (AAVC7) with a capsid sequence close to AAV13 was discovered. HEK-293 high-titer producible vectors with cloned AAVC7 and AAV9 were first assessed for in vivo biodistribution analysis with a CMV-YFP reporter cassette in mice (n=6 animals). 2 weeks after intravenous (i.v.) injection (per vector 5E12 vector genome copies(vgc)/kg), AAVC7 equaled AAV9’s excellent cardiac transduction and YFP mRNA expression abilities by vgc and mRNA levels in left ventricles (LV). In contrast to AAV9s significantly higher hepatic than cardiac enrichment, AAVC7 was almost undetectable in liver and other organs (e.g. brain) with a 1000-fold higher heart/liver (h/l) vgc ratio than AAV9. AAVC7’s biodistribution was subsequently compared to AAV9 in mice using a cardiac troponin (cTnT)-YFP reporter system at a clinically relevant dosage (5E13 vgc/kg; n=6 animals). 2 weeks after i.v. injection, AAV9 induced high dose-dependent hepatic YFP protein IHC and IB expression. In contrast, AAVC7 presented a homogeneous YFP protein expression in cardiomyocytes while YFP protein expression was absent in liver. Given its high cardiac specificity, AAVC7 with the cTNT-relaxin family peptide receptor 1 (rfxp) inotropic response gene cassette, which can be acutely activated using the peptide ligand relaxin (rel), was injected into mice (1E14 vgc/kg; n=6 animals). After 2 weeks, the treated murine hearts showed cardiac-restricted rfxp expression and significant increase in cardiac contractile performance by LV catheterization in response to 50 µg single intraperitoneal (i.p.) rel injection. Subsequent comparison of i.v. delivered AAVC7 and AAV9 in farm pigs (1E11 vgc/kg, n=3 animals), each with a CMV-YFP reporter system, confirmed the high cardiac specificity of AAVC7 over AAV9 (>1000-fold) and homogeneous LV and RV transduction of the human-sized hearts. In human (h)iPSC-cardiomyocyte-EHTs and hiPSC-hepatocytes, the ability of AAVC7 (MOI 1E4 vgcs) to homogenously transduce a human myocardial tissue equivalent and avoid hepatocyte transduction was validated using YFP protein expression analysis via anti-YFP immunofluorescent microscopy and benchmarked against AAV6.

Conclusion:
AAVC7, discovered in human failing myocardium, combines the clinically desirable properties of high and homogeneous in vivo transduction of murine and pig human-sized hearts with excellent liver detargeting after systemic administration and producibility. Such characteristics may destine rAAVC7 a suitable vector for safe and effective human cardiac GT.