PROJECT SUMMARY Our laboratories have developed novel techniques for the editing B-cell receptors of human primary B cells. Using newly identified CRISPR/Cas proteins and innovative homology-directed repair templates, we can efficiently overwrite the endogenous variable heavy (VH) and variable light (VL) segments of a mature VDJ- recombined BCR with the variable genes of broadly neutralizing HIV antibodies (bNabs). Importantly, these variable genes are placed in their respective natural loci, and – excepting the new VH and VL segments – these edited B cells are indistinguishable from unmodified mature, naïve B cells. We describe these edited B cells as “chimeric antigen receptor B cells”, or CAR B cells, evoking the more familiar CAR T cells. These CAR B cells replicate, differentiate, affinity mature, and secrete antibodies in vivo providing an efficient delivery vehicle for bNabs. CAR B cells represent a key advance over passive infusion or gene therapy delivery of bNabs, because they do not raise anti-drug antibodies against their novel BCR, and because the can affinity mature in response to an antigen, including HIV-1 emerging from a reactivated reservoir. They can thus adapt in real time to the specific viral variants in the reservoir. To date, however, we have only transformed CAR B cells ex vivo by isolating primary B cells from a particular host, transforming them by electroporation of CRISPR/Cas RNPs and DNA repair templates, and re-infusing the CAR B cells into the host. Although ex vivo CAR B transformation could be clinically viable, it would likely be a prohibitively expensive procedure for most HIV-positive persons. Here we propose to perform the CAR B transformation procedure in vivo by developing a B cell-tropic adeno-associated virus (AAV)-based gene therapy vector and CRISPS/Cas editing cassette that could be administered intravenously; a comparatively fast and inexpensive procedure. This proposal is divided into three aims. In Aim 1, we draw upon our extensive experience modifying AAV capsids to target specific cell types to create a B cell-tropic capsid. In Aim 2, we develop and evaluate a range of AAV-delivered CRISPR/Cas editing cassette designs for their ability to efficiently transform CAR B cells. Lastly, in Aim 3, we optimize an immunogen and immunization strategy to drive proliferation and affinity maturation of newly transformed CAR B cells. These studies will make clinically viable a promising approach for suppressing an established HIV-1 infection or preventing a new one in high-risk persons.