ABSTRACT A variety of gene therapy strategies have been developed to achieve HIV cure. These strategies include genetic methods to render immune cells resistant to infection or to enhance immune effector cell anti-HIV activity. In this latter instance, genetic engineering of B cells has provided a highly novel means to achieve vectored immunotherapy for production of broadly neutralizing anti-HIV antibodies (bnAbs). Of note, the exceptional utilities of gene editing have been successfully employed to achieve precision genome modification of B cells to accomplish this technical end. In this approach, primary mature B cells from the periphery are modified to express HIV bnAbs as functional antigen receptors spliced to cell endogenous heavy chain constant genes. These cells are then expanded and affinity-matured, using vaccines or viral antigen in vivo, resulting in the elicitation of durable, self-tolerant, and isotyped-switched broadly neutralizing antibodies and memory. Here-to-fore this promising B cell approach has mandated ex vivo genetic modification to practically accomplish effective cell engineering. This facet of the strategy thereby entrains technical and methodological complexities practically limiting this approach. Clearly, the ability to accomplish genetic modification of B cells in vivo would render this approach of greater applicability and accessibility. Further to this end, the ability to achieve such in vivo B cell transduction, in an efficient and selective manner, would be key to realizing the benefits of this technology with an acceptable margin of safety. To this end, we have explored the utilities of replication defective adenoviral vectors (Ad) to address the mandates for in vivo transduction of B cells. In this regard, we have developed a novel “triple targeting” approach that allows efficient and selective gene delivery to key target cells in vivo. In addition, adenovirus provides a unique framework for effective and economical in vivo delivery of CRISPR/Cas9 and donor DNA for targeted integration of bnAb genes into the B cell genome by homology directed repair in situ. Of additional note, the use of non-human primate Ads allows derivation of vectors that can traverse immune barriers to human adenovirus-based vectors. In the aggregate, these combined facets of adenovirus provide the critical functional capacities allowing us to address the mandates of an in vivo applied anti-HIV B cell vectored immunotherapy strategy.

PROJECT NARRATIVE We will develop a vectored immunotherapy approach for HIV by means of in vivo modification of B cells to express anti-HIV antibodies. To feasibilize this approach we will develop adenoviral vectors capable of efficient and selective transduction of target B cells in vivo.