926. Recombinant AAV Vectors Pseudotyped with Serotype-1 Capsid Mediates Early Onset of Gene Expression in Mouse Hearts

Top of pageAbstract Adeno-associated virus serotype 2 (AAV2) has been used most frequently as vector in experiments on cardiac gene therapy. We have used this vector to deliver vascular endothelial growth factor (VEGF) gene controlled by the hypoxia response element (HRE) and a cardiac myosin light chain 2v promoter. We achieved cardiac-specific and hypoxia-responsive gene expression that induced neovascular formation in ischemic murine hearts and improved cardiac function. However, gene expression mediated by AAV2 vector in hearts was delayed and did not reach the maximum level until about 4 weeks after intramyocardial injection. Irreversible myocardial injury could have occurred during this period of delay. Because hypoxia inducible factor 1 (HIF-1) is induced immediately at the onset of hypoxia, earlier onset of the HRE regulated gene expression should be more effective in inducing therapeutic angiogenesis. Recently, studies on AAV with different serotypes have shown that they differ in their transduction efficiency with different tissues. To identify a serotype that can efficiently mediate early expression of transgene in the myocardium, we packaged into serotypes 1 to 5 capsids, AAV vector carrying CMV promoter driving the mouse erythropoietin (Epo) gene. The packaged vectors were injected into murine hearts intramyocardially. Gene expression was studied by real-time RT-PCR. The kinetics of Epo expression was studied by measuring the hematocrit after AAVEpo gene transduction at different time points. We found that AAV1, 4 and 5 mediated earlier and higher gene expression than AAV2. Gene expression mediated by AAV1 increased faster than AAV4 and 5, and became the highest after day 4. To compare the early gene expression mediated by AAV serotype 1 and 2, we packaged into these capsids AAV-LacZ vectors that carried a CMV promoter driving LacZ gene expression and injected them to normal and ischemic hearts. Gene expression was detected by LacZ staining. The LacZ expression was detected in the normal and ischemic myocardium one day after AAV1-LacZ injection, while no LacZ expression could be detected in the hearts injected with AAV2-LacZ at this time. At day 14, 100% of the cardiomyocytes around needle injection sites were infected by AAV1-LacZ vector, whereas only sporadic cardiomyocytes were infected by AAV2-LacZ. These results indicate that AAV1 can mediate gene expression within one day. HRE regulated VEGF gene packaged in this capsid will induce very early gene expression and therefore enhance the therapeutic effect.