Engineering Adeno-associated virus vectors for the in vivo generation of CAR T cells targeting HIV B cell reservoirs

About This Grant

Project Summary There are currently over 40 million people living with human immunodeficiency virus (HIV) worldwide. The gold standard treatment for HIV is antiretroviral therapy (ART) which, while capable of suppressing viremia, is unable to achieve viral clearance meaning that the therapy must be taken lifelong. Recently, cytotoxic T cells reprogrammed to express a chimeric antigen receptor (CAR) that targets the HIV envelope protein have emerged as a new treatment strategy that can potentially mediate HIV clearance through seeking out and lysing HIV infected cells. However, the cost associated with the generation of CAR T cells is prohibitively expensive due to extensive ex vivo manipulation steps performed in specialized facilities. In vivo generation of CAR T cells could substantially reduce the cost of therapy but must meet two requirements: (I) the CAR construct must be delivered specifically to T cells and (II) the CAR construct must be integrated into the genome to ensure persistence of the therapy. These two requirements are difficult to achieve with currently available in vivo gene delivery vehicles as they require the ability to target specific cells and perform off target free genome editing. Adeno-associated virus (AAV) is a leading platform for in vivo gene delivery, with a proven clinical track record, but it is constrained by broad tissue tropism and the lack of precise, off-target-free integration methods. To address these limitations, this project leverages two recent innovations from our laboratory: (1) programmable retargeting of AAV capsids through covalent linkage to antibodies or nanobodies, and (2) packaging of circular RNA (circRNA) inside AAV particles to transiently deliver gene editing tools such as Streptococcus pyogenes Cas9 (spCas9). We hypothesize that T cell-directed delivery of circRNA encoding spCas9, in combination with a homology-directed repair (HDR) template, will enable efficient and specific reprogramming of T cells into anti- HIV CAR T cells. In Aim 1, we will validate this approach by testing whether circRNA-AAV vectors encoding Cas9 and an HDR template can reprogram primary human T cells ex vivo into CAR T cells capable of targeting HIV- infected cells. In Aim 2, we will develop an induced pluripotent stem cell (iPSC) line lacking the canonical AAV receptor (AAVR), then generate T cells from this line to systematically investigate whether T cell-enriched receptors can support AAV transduction in the absence of AAVR. Identifying such alternative receptors would provide a foundation for engineering capsid-receptor interactions tailored specifically to T cells. The proposed studies are expected to (1) establish an AAV-based platform for site-specific genome integration via circRNA-encoded Cas9, (2) demonstrate efficient and safe generation of CAR T cells targeting HIV-infected cells, (3) uncover T cell-specific receptors that can mediate AAV entry, and (4) define receptor properties that enable functional transduction independent of AAVR. Together, these outcomes will advance the long-term goal of enabling in vivo generation of therapeutic CAR T cells while providing broadly applicable tools for precision AAV retargeting.