Bipolar CD4-targeted dual-DARPin-55/57 lipid nanoparticle enables efficient CRISPR/Cas-mediated HIV-1 DNA excision and reactivation blockade in latent CD4 T cell lines.

The persistence of HIV-1 latent reservoirs remains the principal barrier to a cure, as viral rebound occurs upon interruption of antiretroviral therapy. CRISPR/Cas genome editing offers a promising strategy to excise proviruses from host genome; however, the absence of a targeted and clinically viable delivery platform has hindered its translational application. Here, we report a chemistry-driven, CD4-targeted lipid nanoparticle (LNP) delivery platform employing a unique bipolar conjugation strategy to decorate dual CD4-targeted Designed Ankyrin Repeat Proteins (DARPins-55 and -57) on LNP (dual-DARPin-LNP). The N- and C-terminally modified DARPin-55/57 was thiolated stepwise, then bipolar maleimide-thiol coupling conjugated the thiolates to the maleimide-functionalized LNP surface. This coupling strategy ensured DARPin proper orientation on the LNP surface for efficient uptake by resting CD4 T cells. This dual-DARPin-LNP system was engineered for selective and efficient co-delivery of spCas9-GFP mRNA (Sp9m) and HIV-1-specific single-guide RNAs (sgRNAs) targeting LTR and Gag (LGsg) into HIV-1 latently infected CD4 T cells. In widely used HIV-1 latency models with defined proviral modifications (J-Lat 10.6 and 2D10 cell lines), dual-DARPin-LNP loaded with Sp9m/LGsg efficiently excised integrated HIV-1 proviral DNA, as confirmed by standard PCR genotyping, absolute digital PCR quantification, confocal microscopy, and flow cytometry. Importantly, proviral excision functionally blocked HIV-1 reactivation following stimulation with latency-reversing agents suberoylanilide hydroxamic acid (SAHA) and TNFα. Together, these findings establish a modular, non-viral, receptor-guided delivery platform for CD4 T cell targeting and provide proof-of-concept for precise HIV-1 DNA excision and reactivation blockade in established latency models. This new strategy represents a step toward next-generation curative interventions against persistent HIV-1 infection.