PROJECT SUMMARY We have developed what appears to be an effective way to prevent HIV-1 infection despite high-dose intravenous challenges. This approach combines eCD4-Ig, an exceptionally broad and potent HIV-1 entry inhibitor with an adeno-associated virus (AAV) delivery system. After a one-time intramuscular inoculation with AAV-eCD4-Ig, rhesus macaques were protected for more than a year from multiple high-dose challenges with SHIV-AD8 and SIVmac239. This protection is likely to last for several additional years or more. A similar approach might also be taken using a cocktail of AAV-expressed broadly neutralizing antibodies (bNAbs), presuming that the problem of their greater immunogenicity can be addressed. In short, we are close to providing effective, long term protection for high-risk individuals such as intravenous drug users. However, there is one clear hurdle remaining. Specifically, while it is likely that eCD4-Ig and bNAbs are safe when passively administered, these proteins have never been expressed in many people for long periods of time, and there is no easy remedy for an adverse event. To that end, we are intensively exploring ways to suppress or stop entirely expression of an AAV transgene. We propose here to develop a number of small on- and off-switches that modulate transgene message-RNA stability, as well as kill-switches that permanently halt transgene expression. Our focus is on switches that are practical for in vivo use, always keeping our eye on the larger goal of making an effective HIV-1 prophylaxis strategy safe.