PROJECT SUMMARY/ABSTRACT
Modification of autologous T cells with chimeric antigen receptor (CAR) molecules was first proposed nearly 30
years ago as a therapy for people living with HIV. Since then, CAR-T cells have emerged as a potent and highly
successful therapy for liquid tumors, while HIV-specific CAR-T cells have only begun to show efficacy in large
animal models and clinical trials. Based on our longstanding interest in CAR-T cell therapies for HIV, we posit
three primary barriers that limit the curative potential of this approach. First, low levels of HIV-1 antigen at the
cell surface (namely Env protein), especially during antiretroviral therapy (ART), render latently infected cells
nearly invisible to CAR-T cells and other virus-specific immune effectors. Second, the wealth of knowledge
regarding cellular trafficking of the viral Env protein has yet to be thoroughly applied in the context of Env-
dependent HIV cure strategies. Third, CAR-T cell persistence and function wane over time, prior to complete
clearance of the latent HIV reservoir. Our groundbreaking preliminary data outlines a path to overcome these
limitations. We recently reported findings in four rhesus macaques that were infected with an HIV-like virus,
suppressed by ART and then infused with virus-specific CAR-T cells containing the CD4 extracellular domain
(CD4CAR). To expand these potent antiviral effectors in vivo, animals were next boosted with an irradiated cell
line stably expressing HIV-1 Env. Following ART treatment interruption (ATI), viral control was observed in 2 of
4 animals, consistent with robust and Env-dependent expansion of CD4CAR-T cells. The central goal of this
proposal is to increase the potency and feasibility of this approach. In AIM 1, we will transition our Env boosting
strategy from an immortalized cell line to an FDA-approved mRNA lipid nanoparticle (mRNA-LNP) platform,
analogous to the Moderna and Pfizer/BioNTech vaccines for SARS-CoV-2. Env immunogens will be optimized
for CD4CAR T cell interactions and developed as Env mRNA-LNP vaccines. In AIM 2, we will use CRISPR-
Cas9 gene editing to extend the durability and function of CD4CAR-T cells. We will compare a series of CAR
products that carry inactivated immune checkpoint alleles, which we hypothesize will support more durable
function and efficiently clear persistent viral reservoirs. In AIM 3, we will benchmark Env mRNA-LNP and immune
checkpoint gene editing strategies in our vetted nonhuman primate (NHP) model of HIV gene therapy. These
experiments will feature a powerful competitive repopulation study design, providing critical information on basic
CAR biology that cannot be gathered in clinical studies. Together, these aims build on what we believe to be the
most promising anti-HIV cell and gene therapy approach reported to date. Our unique and highly informative
NHP model of HIV persistence and CAR-T cell therapy will fill in critical gaps in knowledge regarding CAR-T cell
safety and function in limited antigen environments, and facilitate clinical translation both in developed and
developing nations. The lessons we learn from these studies will be applicable not only as a curative therapy for
HIV-1, but for a range of diseases such as solid tumors where CAR-T cell therapies must be similarly augmented.