Research Department Cell and Gene Therapy, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.
German Centre for Infection Research (DZIF), partner site, Hamburg, Germany.
Gene Ther. 2021 Sep;28(9):572-587. doi: 10.1038/s41434-021-00259-5. Epub 2021 Apr 19.
Ex-vivo gene editing in T lymphocytes paves the way for novel concepts of immunotherapy. One of those strategies is directed at the protection of CD4-T helper cells from HIV infection in HIV-positive individuals. To this end, we have developed and optimised a CCR5-targeting TALE nuclease, CCR5-Uco-hetTALEN, mediating high-efficiency knockout of C-C motif chemokine receptor 5 (CCR5), the HIV co-receptor essential during initial infection. Clinical translation of the knockout approach requires up-scaling of the manufacturing process to clinically relevant cell numbers in accordance with good manufacturing practice (GMP). Here we present a GMP-compatible mRNA electroporation protocol for the automated production of CCR5-edited CD4-T cells in the closed CliniMACS Prodigy system. The automated process reliably produced high amounts of CCR5-edited CD4-T cells (>1.5 × 10 cells with >60% CCR5 editing) within 12 days. Of note, about 40% of total large-scale produced cells showed a biallelic CCR5 editing, and between 25 and 42% of produced cells had a central memory T-cell phenotype. In conclusion, transfection of primary T cells with CCR5-Uco-hetTALEN mRNA is readily scalable for GMP-compatible production and hence suitable for application in HIV gene therapy.
体外基因编辑 T 淋巴细胞为免疫疗法的新概念铺平了道路。其中一种策略旨在保护 HIV 阳性个体中的 CD4-T 辅助细胞免受 HIV 感染。为此，我们开发并优化了一种靶向 CCR5 的 TALE 核酸酶，即 CCR5-Uco-hetTALEN，可介导 C-C 基序趋化因子受体 5（CCR5）的高效敲除，该受体是初始感染期间 HIV 的共受体。该敲除方法的临床转化需要根据良好生产规范（GMP）将制造过程扩大到临床相关数量的细胞。在这里，我们提出了一种 GMP 兼容的 mRNA 电穿孔方案，用于在封闭的 CliniMACS Prodigy 系统中自动生产 CCR5 编辑的 CD4-T 细胞。该自动化过程可在 12 天内可靠地产生大量 CCR5 编辑的 CD4-T 细胞（>1.5×10^6 个细胞，CCR5 编辑率超过 60%）。值得注意的是，大约 40%的总大规模生产的细胞显示出双等位基因 CCR5 编辑，并且 25%至 42%的产生的细胞具有中央记忆 T 细胞表型。总之，用 CCR5-Uco-hetTALEN mRNA 转染原代 T 细胞可轻松扩大规模，以进行 GMP 兼容生产，因此适用于 HIV 基因治疗。
