Department of Therapeutic Radiology and Genetics, Yale University School of Medicine, New Haven, Connecticut, USA.
Mol Ther Nucleic Acids. 2013 Nov 19;2(11):e135. doi: 10.1038/mtna.2013.59.
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.Molecular Therapy-Nucleic Acids (2013) 2, e135; doi:10.1038/mtna.2013.59; published online 19 November 2013.
可生物降解的聚（乳酸-共-乙醇酸）（PLGA）纳米颗粒（NPs）包载三链形成肽核酸（PNAs）和供体 DNA，用于 CCR5 基因的重组介导编辑，被合成用于递送至人外周血单核细胞（PBMCs）。含有 CCR5 靶向分子的 NPs 能够以低细胞毒性高效进入 PBMCs。深度测序显示，单次处理该制剂可使 CCR5 基因的靶向频率达到 0.97%，而 CCR2 基因的脱靶频率仅为 0.004%，差异达 216 倍。NP 处理的 PBMCs 有效地植入免疫缺陷 NOD-scid IL-2rγ（-/-）小鼠，并且在移植后 4 周在脾淋巴细胞中检测到靶向 CCR5 修饰。在感染 R5 嗜性 HIV-1 后，用 CCR5-NP 处理的 PBMCs 移植的人源化小鼠与用模拟处理的 PBMCs 移植的对照小鼠相比，CD4(+)T 细胞水平显著升高，血浆病毒 RNA 载量显著降低。这项工作证明了 PLGA-NP 包封的 PNA 基基因编辑分子用于靶向修饰人 PBMCs 中的 CCR5 的可行性，作为赋予 HIV-1 抗性的平台。分子治疗-核酸（2013 年）2，e135；doi:10.1038/mtna.2013.59；在线发表 2013 年 11 月 19 日。
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