Efficient Clinical Scale Gene Modification via Zinc Finger Nuclease-Targeted Disruption of the HIV Co-receptor CCR5
Efficient Clinical Scale Gene Modification via Zinc Finger Nuclease-Targeted Disruption of the HIV Co-receptor CCR5
复制标题
DOI：
10.1089/hum.2012.172
复制
发表时间：
2013-03-01
影响因子：
4.2
通讯作者：
Levine, Bruce L.
中科院分区：
文献类型：
--
作者：
Maier, Dawn A.;Brennan, Andrea L.;Levine, Bruce L.
由于 HIV 需要 CD4 和辅助受体（最常见的是 C-C 趋化因子受体 5 (CCR5)）才能进入细胞，因此靶向 CCR5 表达是治疗 HIV 感染的一种有吸引力的方法。用特异性破坏趋化因子受体 CCR5 编码序列的锌指蛋白核酸酶 (ZFN) 处理 CD4(+) T 细胞，可在体外和体内诱导对 HIV 感染的抵抗力。编码 CCR5-ZFN 的嵌合 Ad5/F35 腺病毒载体允许在 T 淋巴细胞的抗 CD3/抗 CD28 共刺激后有效递送和瞬时表达。我们提供的数据显示，与报道的 T 淋巴细胞 Ad5/F35 转导方法相比，CD3/CD28 共刺激显着提高了转导效率。对实验室规模工艺的修改，结合临床兼容的试剂和方法，产生了强大的离体制造工艺，能够从健康和 HIV+ 供体中产生 > 10 10 CCR5 基因编辑的 CD4+ T 细胞。 ZFN 修饰细胞和对照细胞之间的 CD4+ T 细胞表型、细胞因子产生和全部功能相当。在咨询监管机构后，我们进行了体内毒性研究，结果显示没有检测到 ZFN 特异性毒性或 T 细胞转化。基于这些发现，我们启动了一项临床试验，测试 CCR5 基因编辑的 CD4+ T 细胞移植在 HIV-1 感染研究对象中的安全性和可行性。
Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4(+) T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating > 10 10 CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection.