Targeted Delivery of CRISPR Cas12a RNPs for HIV Gene Editing - Despite effective suppression of HIV by antiretroviral therapy (ART), a cure remains elusive because HIV persists in long-lived, latently infected CD4⁺ cell reservoirs that rapidly reignite infection upon treatment interruption. Eliminating these reservoirs is the primary barrier to achieving a functional HIV cure. CRISPR–Cas12a ribonucleoproteins (RNPs) provide a potent genome-editing strategy, enabling precise excision of essential proviral regions, multiplex targeting, and transient, high-specificity activity. We previously developed Cas12a gRNAs targeting conserved HIV sequences with no predicted human off-targets and demonstrated that Cas12a RNPs targeting the highly conserved Nef/3′-LTR region potently inhibited HIV, comparable to validated Cas9 controls. However, clinical translation is limited by the lack of safe and effective delivery systems capable of selectively reaching HIV-infected CD4⁺ cells in lymphoid tissues and enabling efficient cellular uptake and endosomal escape. Lipid nanoparticles (LNPs) represent the most clinically validated non-viral delivery platform, with proven safety and efficacy in multiple FDA-approved siRNA and mRNA therapeutics. We hypothesize that the challenges of targeted CRISPR–Cas12a delivery can be overcome by rational, machine-learning–guided engineering of LNP formulations to enable selective delivery of active Cas12a RNPs to CD4⁺ cells in lymphoid tissues, thereby eliminating persistent viral reservoirs. This study is innovative in integrating lymphoid-tropic, machine-learning–designed LNPs— addressing the intrinsic hepatic bias of conventional LNPs—with CD4-targeted delivery of pre-assembled Cas12a RNPs to achieve precise excision of latent HIV provirus. By combining extrahepatic biodistribution, receptor- mediated CD4⁺ cell targeting, efficient endosomal escape, and compact multiplex Cas12a genome editing, this strategy overcomes key limitations of existing CRISPR-based HIV therapies. By applying this innovative strategy, we aim to develop and establish a highly precise, safe, and clinically translatable platform toward a functional HIV cure. Specifically, in Aim 1, we will apply machine-learning–guided design to develop and optimize lymphoid-tropic LNPs encapsulating functional Cas12a RNPs and surface-functionalized with a CD4 receptor–binding peptide to ensure selective binding to CD4⁺ cells. In vitro studies in HIV-infected cell lines will evaluate CD4-specific uptake, intracellular delivery, and Cas12a-mediated genome-editing efficiency. In Aim 2, we will evaluate the in vivo performance of CD4-targeted Cas12a RNP–LNPs by assessing lymphoid tissue biodistribution, selective uptake by CD4⁺ cells, and antiviral efficacy in humanized HIV-infected mice. We will determine whether systemically administered LNPs preferentially accumulate in lymphoid organs, deliver Cas12a RNPs into CD4⁺ cells, achieve on- target editing of the HIV Nef/3′-LTR region, and suppress viral gene expression, p24 production, and infectious virus release. This study directly targets latent HIV in CD4⁺ cell reservoirs using a clinically translatable, non-viral CRISPR– Cas12a RNP–LNP platform, with the potential to move HIV treatment beyond lifelong viral suppression toward a durable functional cure.
