Targeted Inactivation of the CCR5 Gene via PNA-induced Homologous Recombination

Peptide nucleic acids (PNAs) bind duplex DNA in a sequence-specific manner, creating triplex structures that can provoke DNA repair and produce genome modification. CCR5 encodes a chemokine receptor required for HIV-1 entry into human cells, and individuals carrying mutations in this gene are resistant to HIV-1 infection. Transfection of human cells with PNAs targeted to the CCR5 gene, plus donor DNAs designed to introduce stop codons mimicking the naturally occurring CCR5-delta32 mutation, produced 2.46% targeted gene modification. CCR5 modification was confirmed at the DNA, RNA and protein levels and was shown to confer resistance to infection with HIV-I. Targeting of CCR5 was achieved in human CD34+ hematopoietic stem cells (HSCs) with subsequent engraftment into mice and persistence of the gene modification more than four months post-transplantation. This work suggests a therapeutic strategy for CCR5 knockout in HSCs from HIV-1-infected individuals, rendering cells resistant to HIV-1 and preserving immune system function.;To further enhance the efficiency and reduce the toxicity of gene targeting in primary human hematopoietic cells, we have also recently tested the use of biodegradable poly (lactide-co-glycolide) (PLGA) nanoparticles (PLGA-NPs) that can encapsulate both the triplex-forming PNAs and the donor DNAs for facile delivery into various cell types. CCR5-targeted PLGA-NPs were formulated by a double-emulsion solvent evaporation technique and evaluated for their ability to enter CD34+ HSCs or peripheral blood mononuclear cells (PBMCs) and deliver PNA and donors for gene targeting. Efficient cellular uptake of the PLGA-NPs in both cell types was verified by FACS analysis, and increased targeted modification was seen with a reduction in cellular toxicity compared to conventional nucleofection techniques. PBMCs treated with PLGA-NPs were injected into NOD-scid IL2rgammanull HLA A2.1 adult mice and were able to engraft at levels equal to untreated cells, whereas nucleofected cells showed poor engraftment. The presence of the targeted CCR5 modification was detected in the spleen of the engrafted mice four weeks post-transplantation. This work suggests a potential therapeutic strategy for permanently inactivating the CCR5 coreceptor in human HSCs or PBMCs from HIV-1 infected individuals to create a reservoir of cells that are virus-resistant, thus preserving immune system function.