30. Cardiac-Specific Gene Expression Using Pseudotyped AAV Vectors and the Cardiac Troponin-T Promoter

Abstract

AAV vectors transduce a wide range of tissues in-vivo and provide for long-term gene expression without provoking an immune response. Following intravenous (IV) injection, AAV serotypes 1 & 6 have been shown to provide for transgene expression throughout the body, including cardiomyocytes. However, the ability to transduce a wide range of tissues could be a disadvantage for cardiac gene therapy protocols, since successful gene therapy often depends on restricting transgene expression to the tissue/organ of interest. In the present study, AAV-2 based vectors carrying the firefly luciferase gene under control of the CMV (ACMVLuc) or cardiac troponin-T (AcTnTLuc) promoters were cross-packaged into capsids from AAV serotypes 1 or 6. These vectors were used for evaluating the magnitude, duration and tissue-specificity of gene expression following IV injection into neonates, and following direct injection into the left ventricular (LV) wall of adult mice. For neonatal mice (8 days old), a total of 1E+11 vector particles were injected via jugular vein, and luciferase expression was monitored using in-vivo bioluminescence imaging followed by ex-vivo bioluminescence imaging and in-vitro luciferase assays. Luciferase expression was evident within 3 days after vector injection, and was observed throughout the body in neonates injected with ACMVLuc. Ex-vivo imaging and in-vitro luciferase assays of various organs showed that luciferase expression was highest in muscle (chest wall, skeletal and heart) and liver tissue in mice injected with ACMVLuc packaged into AAV-6 capsids. In contrast, luciferase expression was predominantly restricted to the heart in mice that were injected with AcTnTLuc. Furthermore, the AAV-6 capsid was at least 30-fold more efficient than the AAV-2 capsid for mediating cardiac gene delivery in neonatal mice. Capsids from AAV serotypes 1 & 6 appeared to be equally efficient for mediating cardiac gene delivery after IV injection in neonates. In adult mice, a total of 5E+10 virus particles were injected into the LV wall, and luciferase expression was monitored using in-vivo imaging followed by in-vitro luciferase assays. In mice injected with AcTnTLuc packaged into capsids from AAV serotypes 1 or 6, luciferase expression was evident from day 3 onward and reached a plateau between 1-2 weeks post- injection. Both serotypes 1 & 6 provided for high levels of gene expression that were sustained for at least 45 days. In conclusion, capsids from both AAV serotypes 1 & 6 deliver genes to the heart more efficiently than the commonly used serotype AAV-2, as evidenced by early onset and high-level gene expression in neonates following IV injection and in adult mouse hearts following direct injection into the myocardium. When employed in conjunction with a cardiac-specific promoter (e.g., cardiac troponin-T) gene expression from these highly-efficient serotypes can be largely restricted to the mammalian heart. The use of tissue-specific promoters in combination with highly efficient AAV capsids (e.g., AAV-1 & -6) further enhances the potential of AAV vectors for human gene therapy protocols.