Adeno-associated virus (AAV) 6 provides efficient gene transfer upon intramyocardial injection in vivo

Abstract

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Innovation Council Eurostars Background Cardiac gene transfer utilizing adeno-associated virus (AAV) is a promising approach for the treatment of various cardiovascular diseases. Intramyocardial injection is an efficient delivery route, and is particularly relevant for ischemic disease or pacemaker and conduction system abnormalities, where local treatment is favoured. However, which AAV variants provide efficient cardiac gene transfer after intramyocardial injection remains to be tested. Aim This study aims to compare several cardiotropic AAV variants in order to identify their relative potency in the setting of direct intramyocardial injection. Methods A green fluorescent protein (GFP)-coding sequence was packaged in the two benchmark AAV capsids, AAV6 and AAV9, and a selection of recently reported AAV9 capsid variants which are named MyoAAVs. These AAV vectors were added to neonatal rat ventricular cardiomyocytes (NRVM) and their transduction efficiency was evaluated by flow cytometry and quantitative PCR. AAV6, AAV9 and MyoAAV4A vectors were then delivered into the left ventricle of FVB mice via intramyocardial injection. AAV6 and AAV9 carrying GFP in a different expression cassette were also intramyocardially injected in FVB and Bl6 mice. Direct fluorescence, transgene mRNA, and viral genomes were quantified to determine the transduction efficiency 4 weeks post injection. Picrosirius red staining was used to assess whether AAV-mediated GFP delivery causes cardiac fibrosis. We further studied GFP encoding AAV6 and AAV9 vectors upon intramyocardial injection into the left ventricle of pigs and determined the transduction efficiency by quantitative PCR. Results NRVM transduction experiment revealed that among the tested capsid variants, AAV6 is the most efficient in transducing cardiomyocytes, followed by MyoAAV4A. In mice, AAV6 is most effective in transducing myocardium upon intramyocardial injection, resulting in approximately 10-fold higher transgene expression than that obtained with AAV9 vector and 2-fold higher than that with MyoAAV4A vector. Histology revealed no vector-related cardiac fibrosis. The use of different expression cassettes and animal strains consistently showed that AAV6 outperforms AAV9 in cardiac transduction efficiency. Transgene expression in liver was only observed in Bl6 mice injected with AAV9. In porcine myocardium, AAV6 led to robust transgene expression after intramyocardial injection and this expression level is higher than that of AAV9. Conclusion Among the AAV variants tested, AAV6 appears to be the most efficient for cardiac transduction by intramyocardial injection. These results suggest that AAV6 represents an attractive vector for direct cardiac gene transfer and for future capsid engineering in the context of local transgene delivery.