Medical School, Department of Pediatrics, Division of Blood and Marrow Transplant & Cellular Therapy, University of Minnesota, Minneapolis, MN 55455, USA.
Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.
Mol Ther. 2022 Jan 5;30(1):130-144. doi: 10.1016/j.ymthe.2021.10.026. Epub 2021 Nov 2.
Disruption of CCR5 or CXCR4, the main human immunodeficiency virus type 1 (HIV-1) co-receptors, has been shown to protect primary human CD4 T cells from HIV-1 infection. Base editing can install targeted point mutations in cellular genomes, and can thus efficiently inactivate genes by introducing stop codons or eliminating start codons without double-stranded DNA break formation. Here, we applied base editors for individual and simultaneous disruption of both co-receptors in primary human CD4 T cells. Using cytosine base editors we observed premature stop codon introduction in up to 89% of sequenced CCR5 or CXCR4 alleles. Using adenine base editors we eliminated the start codon in CCR5 in up to 95% of primary human CD4 T cell and up to 88% of CD34 hematopoietic stem and progenitor cell target alleles. Genome-wide specificity analysis revealed low numbers of off-target mutations that were introduced by base editing, located predominantly in intergenic or intronic regions. We show that our editing strategies prevent transduction with CCR5-tropic and CXCR4-tropic viral vectors in up to 79% and 88% of human CD4 T cells, respectively. The engineered T cells maintained functionality and overall our results demonstrate the effectiveness of base-editing strategies for efficient and specific ablation of HIV co-receptors in clinically relevant cell types.
阻断 CCR5 或 CXCR4（人类免疫缺陷病毒 1 型（HIV-1）的主要共受体）已被证实可保护原代人 CD4 T 细胞免受 HIV-1 感染。碱基编辑可在细胞基因组中引入靶向点突变，因此可以通过引入终止密码子或消除起始密码子而无需双链 DNA 断裂来有效地使基因失活。在这里，我们应用碱基编辑器在原代人 CD4 T 细胞中分别和同时阻断这两个共受体。使用胞嘧啶碱基编辑器，我们观察到高达 89%的 CCR5 或 CXCR4 等位基因中存在提前终止密码子的引入。使用腺嘌呤碱基编辑器，我们在高达 95%的原代人 CD4 T 细胞和高达 88%的 CD34 造血干细胞和祖细胞靶等位基因中消除了 CCR5 的起始密码子。全基因组特异性分析显示，碱基编辑引入的脱靶突变数量很少，主要位于基因间或内含子区域。我们表明，我们的编辑策略可分别防止高达 79%和 88%的人 CD4 T 细胞被 CCR5 嗜性和 CXCR4 嗜性病毒载体转导。经工程改造的 T 细胞保持了功能，总的来说，我们的结果表明碱基编辑策略在临床相关细胞类型中有效且特异性地消除 HIV 共受体的有效性。
