Abstract/Summary People living with HIV (PLWH) continue to harbor virus-infected cells despite suppression by antiretroviral therapy. These reservoir cells are the source of virus rebound that typically occurs in 1-8 weeks if treatment is discontinued. Eliminating the provirus integrated in the genome of infected cells or the infected cells themselves can cure PLWH. The recently discovered genome editing technique called CRISPR is being developed as an approach to target and excise HIV DNA sequences present in infected cells. Large challenges to using the CRISPR approach for HIV elimination are the wide diversity of viral isolates in PLWH and the risk of genotoxicity as the Cas9 enzyme causes double-stranded breaks (DSBs) in the host chromosome. The absence of a targeted delivery system for cells that make up the HIV reservoir increases these risks due to off-target effects. This R01 application in response to RFA-AI-20-076: “New Technologies for the In vivo Delivery of Gene Therapeutics for an HIV Cure” addresses the challenges posed by CRISPR-based gene therapy. We will use a designer recombinase, Brec1, that has been developed to solely target HIV-1 provirus sequences and remove these from the genome. 90% of the major HIV-1 subtype groups A, B, and C are expected to harbor the precise Brec1 target sequence. Brec1 shows no immunogenic potential, does not induce cellular toxicity or display off- target activity. We will in adition re-engineer Brec1 to generate a super-repressor called Brec1-Off that can durably silence HIV transcription by epigenetically modifying HIV promoter sequences. Key to the success of this approach is our development of capsid-engineered adeno-associated viral (AAV) vectors to deliver Brec1 and Brec1-Off specifically to human T cells and myeloid cells in peripheral tissues and the central nervous system. AAV vectors are one of safest clinical gene therapy vectors that are approved for human use, albeit one obstacle is their unselective delivery. We therefore redirect AAV tropism to the human immune cells in tissue compartments which can harbor HIV-1 proviruses by deleting their natural receptor binding and engineering specific target cell binders to their surface. Thus we pave the way to the development of a viable, non-invasive approach for a gene therapy-based cure for HIV/AIDS. The proposal has two specific aims- In Specific Aim 1, we will develop AAV6 and AAV9 vectors with capsids modified to target human CD4 T cells and myeloid cells and optimize their design for expression and delivery of Brec1/Brec1-Off to these human immune cells as well as thoroughly assay AAV-Brec1 interventions in cell models of HIV-1 infection. In Specific Aim 2, we will test the capsid-engineered AAV vectors packaging Brec1 and Brec1-Off in three different physiologically relevant humanized mouse models for HIV infection to assess their ability to inactivate peripheral and CNS reservoirs of HIV-1 that also contain the diverse HIV isolates ...