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…

The National Institute of Allergy and Infectious Diseases awarded East Tennessee State University $569,903 on September 1, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to develop targeted delivery of CRISPR Cas12a ribonucleoproteins for HIV gene editing.

The project addresses persistent HIV reservoirs in latently infected CD4 cells that survive antiretroviral therapy and reignite infection upon treatment interruption. The research integrates machine-learning-guided engineering of lymphoid-tropic lipid nanoparticle formulations with CD4-targeted delivery of pre-assembled Cas12a ribonucleoproteins to enable precise excision of latent HIV provirus from infected cells in lymphoid tissues. The approach combines extrahepatic biodistribution, receptor-mediated CD4 cell targeting, efficient endosomal escape, and compact multiplex Cas12a genome editing to overcome existing limitations in CRISPR delivery.

East Tennessee State University is the recipient and place of performance in Johnson City, Tennessee. The award runs through August 31, 2029. This is a Project Grant, an assistance type for discrete, time-limited research projects under NIH's competitive review framework, evaluated on scientific merit, technical adequacy of experimental design, investigator competency, facility adequacy, budget justification, and relevance to program objectives.

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.