Current antiretroviral therapies suppress HIV replication but fail to eliminate integrated proviral DNA in long-lived CD4+ cells, precluding a cure. CRISPR-Cas9 offers potential for HIV eradication but efficient and cell-specific delivery into HIV target cells remains a major hurdle. We developed CD4-directed Nanoblades (CD4-NBs), murine leukemia virus-like particles pseudotyped with anti-CD4 nanobodies and a fusogenic glycoprotein VSV Gmut, to selectively deliver Cas9-gRNA ribonucleoproteins into CD4+ cells. CD4-NBs selectively delivered cargo to CD4+ cells in vitro and in vivo, achieving efficient gene disruption in primary CD4+ cells. Dual-guide CD4-NBs targeting conserved HIV tat/rev/env regions disrupted proviral DNA, suppressing HIV infection in CD4+ cells. In HIV-infected, ART-pretreated humanized mice, CD4-NBs significantly reduced plasma viremia. While full tissue reservoir clearance was not achieved, repeated dosing did reduce viral RNA and proviral DNA in bone marrow and lungs,
respectively. As such, this proof-of-concept study supports the promise of CD4-NBs as a minimally invasive, CD4+ cell-targeted gene editing strategy for HIV therapy.