Abstract 19439: Adeno-Associated Virus Mediated Gene Delivery: Implications for Scalable in vivo Cardiac Models of Optogenetics
The growing use of adeno-associated viruses (AAVs) as transgene delivery vehicles in clinical trials requires comprehensive assessment of their performance. Their serotype-mediated ability to target specific tissues, as well as their low immunogenicity, make them prime candidates for gene therapy. Here we quantify transgene expression and explore the mechanisms of virus uptake in rat cardiomyocytes (CMs) in vitro and in vivo by the cardiac specific AAV serotypes 1, 6, and 9. In vitro syncytia of neonatal rat ventricular CMs (n=7 per serotype) were infected with AAVs containing the transgene for eGFP. Adult rats (n=6 each serotype) were intravenously injected with 0.1-0.5x1012 AAV particles containing the transgene for channelrhodopsin-2 (ChR2-mCherry). Open chest functional testing of optical excitability was performed on the rat heart in situ 3-4 weeks after infection. In vitro expression profiles revealed that AAV 1 and 6 (6±0.4 and 32±1.7 a.u. respectively) outperformed AAV9, which had no detectable levels of eGFP expression. In contrast, intact hearts expressed significantly higher levels of the transgene using AAV9. Hearts infected at the highest viral titer were optically excitable (4.8 mW/mm2). We further tested the mechanism of virus uptake, focusing on purported receptor-mediators for AAV1/6 (cell surface sialic acid) and AAV9 (the 37/67 kDa lamininR receptor). In vitro desialyzation of the neonatal CMs with neuraminidase treatment (25μL/mL) significantly decreased eGFP expression. We showed robust lamininR expression in the adult rat heart via immunostaining, but no expression in cultured neonatal CMs, in line with the observed differential AAV9 expression. The combined transgene expression and cell surface receptor data explain the preferential efficiency of AAV1/6 in vitro and AAV9 in vivo in the rat heart. Furthermore, AAV9 systemic delivery with a generic promoter resulted in preferred cardiac expression of the delivered opsins.
Author Disclosures: C.M. Ambrosi: None. A. Klimas: None. G. Sadananda: None. E. Entcheva: None.
- © 2016 by American Heart Association, Inc.