PROJECT SUMMARY (Project 4 – A permanent off-switch for AAV transgenes) Adeno-associated virus (AAV)-expressed antibody-like entry inhibitors provide very robust protection from HIV-1 model viruses in rhesus macaques. AAV-mediated expression of broadly neutralizing antibodies or antibody-like molecules can also largely or wholly suppress an established infection in macaques. We thus have an effective vaccine and a pathway to drug-free virologic remission if this same approach could be safely applied to humans. However, there is currently no effective way to inactivate an AAV transgene in case of adverse events, and thus this approach is still considered too risky for most individuals. An effective ‘off-switch’ would have two important uses. First, it would increase the safety of AAV-based vaccines and therapies. Second, it would facilitate eradication studies by allowing sustained expression of a potent antibody or entry inhibitor, and then allowing it to be inactivated so that the rate of viral rebound can be measured. In preliminary data, we show that a morpholino can be used together with a highly efficient ribozyme to induce expression of an AAV transgene. We also show that the Cre recombinase can permanently inactivate an AAV transgene flanked by LoxP sites. Here we proposed to combine these observations to generate a permanent off-switch for an AAV-expressed transgene. We will then demonstrate in mice that this off-switch can halt otherwise efficient expression of two potent HIV-1 entry inhibitors. Finally, we will use the same principles to develop a morpholino-regulated on-switch. We will then use this switch to test whether a 4-month delay in transgene expression from the time of AAV inoculation can limit immune clearance of the transgene. These studies will therefore make AAV-based therapeutics safer, and facilitate study of the impact of long-term antibody expression on the reservoir of latently infected cells.