PROJECT SUMMARY We have developed a novel gene therapeutic approach to achieve an HIV functional cure, wherein broadly neutralizing antibody (bnAb) genes are targeted to the endogenous heavy chain locus in B cells for expression as functional antigen-receptors (BCRs). This takes advantage of the precision of gene editing conferred by CRISPR-Cas9 reagents and homology-directed gene editing. We have shown that these broadly neutralizing BCRs respond to HIV Env immunogens by signaling clonal expansion and germinal center maturation of the engineered cells and thereby generate durable, isotype-switched memory bnAb responses that could be deployed for long-term control of HIV. Given these features of B cell biology, which are preserved by our gene editing approach, we hypothesize that sufficient neutralizing antibody titers in serum could be generated from very few engineered B cells, including the levels achieved by in vivo delivery of genome-editing reagents directly to the target cells. In support of this approach, we have now shown that high-titer bnAb responses can be elicited in mice by vaccination with HIV-Env immunogens when the animals are injected with a transcriptionally-targeted dual-AAV vector system carrying the bnAb editing reagents. Unlike injection of recombinant bnAbs, or AAV- vectored delivery of bnAb-IgG expression cassettes to liver or muscle for long-term secretion from viral episomes, bnAbs generated from in vivo gene-edited B cells in response to vaccination are expected to have the following advantages: 1) be boostable, as needed, in response to viral antigen, 2) express bnAbs as all effector isotypes to expand antiviral immunity, 3) affinity-mature or evolve the bnAb response to keep-pace with a rapidly evolving virus, and 4) be better tolerated by the immune system. To further develop this approach, we have assembled a team that brings together expertise in B cell engineering, vector development, HIV vaccine design, and animal models to further develop this in vivo delivery approach to specifically and efficiently engineer B cells in vivo and to evaluate this approach as an in vivo HIV cure.