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Biodegradable poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) encapsulating triplex-forming peptide nucleic acids (PNAs) and donor DNAs for recombination-mediated editing of the CCR5 gene were synthesized for delivery into human peripheral blood mononuclear cells (PBMCs). NPs containing the CCR5-targeting molecules efficiently entered PBMCs with low cytotoxicity. Deep sequencing revealed that a single treatment with the formulation resulted in a targeting frequency of 0.97% in the CCR5 gene and a low off-target frequency of 0.004% in the CCR2 gene, a 216-fold difference. NP-treated PBMCs efficiently engrafted immunodeficient NOD-scid IL-2rγ-/- mice, and the targeted CCR5 modification was detected in splenic lymphocytes 4 weeks posttransplantation. After infection with an R5-tropic strain of HIV-1, humanized mice with CCR5-NP–treated PBMCs displayed significantly higher levels of CD4+ T cells and significantly reduced plasma viral RNA loads compared with control mice engrafted with mock-treated PBMCs. This work demonstrates the feasibility of PLGA-NP–encapsulated PNA-based gene-editing molecules for the targeted modification of CCR5 in human PBMCs as a platform for conferring HIV-1 resistance.