(191k) Recombinant Adeno-Associated Virus As Gene Delivery Vehicles Targeting the Heart

Gene therapies hold great attention from researchers with the vital role of genetic factors in cardiovascular disease. Adeno-associated virus (AAV) is widely used as a viral vector for gene delivery due to its high efficiency and low immunogenicity. Though AAV has different serotypes preferring specific organs, poor targeting of the heart with a low dose of AAV is a main obstacle to utilizing AAV as a gene delivery vehicle. To achieve high efficiency of gene delivery, we used AAV-DJ which is a hybrid vector originated from a combination of eight AAV serotypes with high transduction efficiency. To target the heart, the specific promoter, cardiac troponin-T (cTnT), was integrated into AAV.

In this study, rAAV-cTnT-YFP was produced from viral productive cells with higher titer and verified in vitro using a human cardiomyocyte cell line, AC16 cells, by RT-PCR and fluorescence imaging. Various doses of rAAV-cTnT-YFP were injected into 7-week-old C57BL/6J mice through retro-orbital injection. Though the liver and spleen showed the transcript levels of the target gene, the heart showed the substantial transcript levels with the dose dependence. The protein expression was confirmed by immunofluorescence merged with the cardiac marker, cTnT. The safety of rAAV-mediated gene delivery was verified by the measurement of alanine transferase assay from serum, echocardiography, and structural change of cardiomyocytes in the tissue. Our rAAV-cTnT-YFP showed the stable expression of the desired gene with targeting to the heart and no safety issues. Based on the data, the dose of rAAV administered should be considered to minimize off-targeting. In future investigations, we generated a postnatal mouse model through subcutaneous injection of rAAV-cTnT-YFP to 7-day-old C57BL/6J to evaluate the difference in the efficiency of gene delivery between adult and postnatal models.