Exosomes as nonviral carrier for targeted delivery of CRISPR-Cas12a for therapeutic HIV-1 proviral DNA editing.

Abstract

Current strategies to treat HIV infection including traditional cART and immunotherapy can effectively suppress viral replication but are unable to eliminate the latent viral reservoir, particularly within circulating immune cells. Although genome editing by CRISPR-Cas provides a promising cure for HIV-1, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we developed an exosome-mediated targeted CRISPR-Cas12a delivery system (EMT-Cas12a), an engineered exosome system enabling targeted delivery of mRNA of Cas12a and crRNAs to CD4+ T cells. The EMT-Cas12a system uniquely optimizes cell-specific targeting, CRISPR-Cas12a expression, crRNAs maturation, nuclear entry efficiency, accuracy cleavage with major delins and achieving dramatically HIV suppression in both cellular and humanized mouse models. Compared with single-crRNA approaches, the multiple crRNA arrays strategy demonstrates enhanced antiviral efficacy in HIV-infected mouse model, ex-vivo-expanded PBMCs from HIV+ subjects and especially in vitro cell lines without detectable HIV DNA. Critically, the system exhibits no detectable off-target effects and restores CD4+ T cell counts in vivo and ex vivo PBMCs, indicating its dual therapeutic potential for viral clearance and immune reconstitution. Altogether, in vitro and in vivo excision of HIV-1 proviral DNA can be achieved via EMT-Cas12a delivery, which could advance efforts toward human clinical trials.