ABSTRACT MarPam Pharma is developing a chimeric antigen receptor (CAR)-T cell treatment that targets viral reservoirs for durable remission of HIV without the need for antiretroviral therapies (ART). This treatment is an HIV-specific CAR (specifically, CD4-MBL-CAR) T cell therapy that employs the CXCR5 chemokine receptor as a homing device to direct anti-HIV killer T cells into “hidden” viral reservoirs in B cell follicles of secondary lymphoid tissue, so that they can kill HIV-producing cells where the majority of viral replication occurs. Using the best in vivo nonhuman primate (NHP) model of HIV, we developed CAR/CXCR5-T cells that show successful homing to B cell follicles, evidence of direct contact with viral RNA+ infected cells, markedly decreased virus in B cell follicles, and decreased viral loads in CAR/CXCR5-T cell treated ART-suppressed Simian immunodeficiency virus- infected NHPs compared to controls. Our human CAR/CXCR5-T cells also show CXCR5-driven migratory and anti-HIV killing characteristics in vitro, showing promise as a treatment for people living with HIV. FDA-approved CAR-T cell therapies are successful in treating various cancers; however, the processes to produce the CAR- T cells are complex, involving leukapheresis and ex-vivo manipulation of peripheral blood mononuclear cells prior to reinfusion back to the patient. Thus, recent efforts are focused on developing universal, off-the-shelf in vivo-produced CAR-T cell therapies, including the use of adeno-associated virus (AAV) gene delivery systems. Since the process to produce an off-the-shelf AAV gene therapy is a fraction of the complexity of ex-vivo production of CAR-T cells individualized for each patient, the expectation is that AAV gene therapy to produce CAR/CXCR5-T cells will eventually prove to be less costly than current life-long therapy for HIV, which is estimated at over $500,000. To develop an off-the-shelf in vivo CAR/CXCR5-T cell product, this proposal seeks to perform the necessary first steps of producing receptor-targeted, AAV-directed CAR-T cells (second generation [2G] -CAR/CXCR5-T cells) in vitro and assessing their targeted efficacy against HIV. Using our intellectual property-protected technology to assemble AAV composites for receptor-targeted gene delivery, we propose in vitro proof-of-concept studies to produce receptor-targeted, AAV-directed 2G-CAR/CXCR5-T cells using AAV composites containing anti-CD5 monoclonal antibody (CD5mab) and carrying a 2G-CAR/CXCR5 DNA payload. As T cells specifically express CD5 on their cell surface, AAV-CD5mab composites carrying 2G- CAR/CXCR5 DNA payload (AAV-CD5mab-2G-CAR/CXCR5) are expected to specifically bind CD5 on T cells and transduce them, thereby producing functional 2G-CAR/CXCR5-T cells that home to B cell follicles in vivo. Successful completion of the proposed studies will lay the groundwork not only for Phase II IND-enabling studies for in vivo HIV 2G-CAR/CXCR5-T cells, but also for other therapeutic ...