Jennifer Sullivan

Adenovirus-mediated expression of p35 prevents hypoxia/reoxygenation injury by reducing reactive oxygen species and caspase activity

OBJECTIVE This study aimed to examine the effects of adenovirus-mediated expression of p35, a baculovirus gene, on apoptosis induced by hypoxia/reoxygenation (H/R) in cardiomyocytes. METHODS Neonatal rat cardiomyocytes were infected with recombinant adenoviral vectors expressing p35 (Ad2/CMVp35) or no transgene (Ad2/CMVEV) and were then subjected to H/R. Separate groups of non-infected cardiomyocytes were treated with pharmacological caspase inhibitors or antioxidants. Cell viability, apoptosis, caspase activity, and cellular reactive oxygen species (ROS) were measured using various assays. RESULTS H/R decreased cell viability and increased cellular ROS levels, caspase activity, and cell apoptosis. Infection with Ad2/CMVp35 effectively inhibited the increase in cellular ROS levels, the activities of caspases 3 and 8, apoptosis, and cell death following H/R, whereas Ad2/CMVEV had no effect. Despite its ability to abolish the increase in caspase activity and partially inhibit apoptosis, the pan-caspase inhibitor ZVAD-fmk (100 microM) failed to significantly reduce cell death induced by H/R. N-acetyl-L-cysteine, an antioxidant, completely inhibited H/R-induced increase in cellular ROS levels, but reduced apoptosis and cell death by 30% only. CONCLUSIONS Adenovirus-mediated expression of p35 effectively inhibits H/R-induced cardiomyocyte apoptosis by reducing cellular ROS levels and inhibiting caspase activity.

Rationally designed AAV2 and AAVrh8R capsids provide improved transduction in the retina and brain

The successful application of adeno-associated virus (AAV) gene delivery vectors as a therapeutic paradigm will require efficient gene delivery to the appropriate cells in affected organs. In this study, we utilized a rational design approach to introduce modifications to the AAV2 and AAVrh8R capsids and the resulting variants were evaluated for transduction activity in the retina and brain. The modifications disrupted either capsid/receptor binding or altered capsid surface charge. Specifically, we mutated AAV2 amino acids R585A and R588A, which are required for binding to its receptor, heparan sulfate proteoglycans, to generate a variant referred to as AAV2-HBKO. In contrast to parental AAV2, the AAV2-HBKO vector displayed low-transduction activity following intravitreal delivery to the mouse eye; however, following its subretinal delivery, AAV2-HBKO resulted in significantly greater photoreceptor transduction. Intrastriatal delivery of AAV2-HBKO to mice facilitated widespread striatal and cortical expression, in contrast to the restricted transduction pattern of the parental AAV2 vector. Furthermore, we found that altering the surface charge on the AAVrh8R capsid by modifying the number of arginine residues on the capsid surface had a profound impact on subretinal transduction. The data further validate the potential of capsid engineering to improve AAV gene therapy vectors for clinical applications.

301. AAV Capsid Engineering to Improve Transduction in Retina and Brain

Gene therapy vectors based on adeno-associated virus (AAV) are currently in clinical studies for numerous disease indications including Lebers congenital amaurosis, age-related macular degeneration, hemophilia, muscular dystrophy and Parkinsons disease. AAV vectors hold considerable promise as therapeutic agents; however there is potential to further improve the efficiency of AAV gene delivery and efficacy by making modifications to the AAV capsid. The AAV capsid can be engineered to incorporate mutations that alter its transduction activity, tropism, biodistribution and immunogenicity. We have constructed variant AAV vectors harboring a variety of capsid modifications including those that negate receptor binding and have tested these vectors in several tissues including the eye and brain. One variant, AAV2HBKO, is an AAV2 based vector containing mutations of critical amino acids known to be required for binding to its receptor, heparin sulfate proteoglycan. Interestingly, an AAV2HBKO vector delivering a secreted transgene, sFLT02, unexpectedly resulted in a 2-log increase in transduction compared to parental AAV2 when delivered subretinally to the mouse eye. Subretinal delivery of an AAV2HBKO vector expressing EGFP demonstrated that these capsid modifications resulted in an increase in photoreceptor transduction compared to the unmodified AAV2 vector. In contrast, the AAV2HBKO vector demonstrated a lack of transduction activity following intravitreal delivery to the mouse eye. In addition, we evaluated the transduction and tropism of AAV2HBKO in the mouse brain. In a head to head comparison with AAV2, the AAV2HBKO vector facilitated widespread striatal and cortical expression following an intrastriatal injection while AAV2-mediated expression was restricted to the site of injection. Similar to AAV2, the tropism of AAV2HBKO was primarily neuronal with little to no transduction of astrocytes or microglia. Biodistribution data suggests that this vector, when delivered systemically in the mouse, has significantly reduced liver transduction but a higher propensity to be delivered to skeletal muscle and heart compared to the wild-type AAV2 vector. We will present data evaluating the transduction activity, tropism and biodistribution of the AAV2HBKO variant. These studies illustrate the potential for improving the efficiency of AAV gene transfer via targeted capsid engineering.