Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
REBIRTH Research Center for Translational Regenerative Medicine, Hannover Medical School, 30625 Hannover, Germany.
Cells. 2020 Sep 29;9(10):2194. doi: 10.3390/cells9102194.
Previous gene therapy trials for X-linked chronic granulomatous disease (X-CGD) lacked long-term engraftment of corrected hematopoietic stem and progenitor cells (HSPCs). Chronic inflammation and high levels of interleukin-1 beta (IL1B) might have caused aberrant cell cycling in X-CGD HSPCs with a concurrent loss of their long-term repopulating potential. Thus, we performed a targeted CRISPR-Cas9-based sgRNA screen to identify candidate genes that counteract the decreased repopulating capacity of HSPCs during gene therapy. The candidates were validated in a competitive transplantation assay and tested in a disease context using IL1B-challenged or X-CGD HSPCs. The sgRNA screen identified () as a potential target to increase HSPC engraftment. Knockout of prior to transplantation was sufficient to induce a selective advantage. Inhibition of p38 increased expression of the HSC homing factor CXCR4 and reduced apoptosis and proliferation in HSPCs. For potential clinical translation, treatment of IL1B-challenged or X-CGD HSPCs with a p38 inhibitor led to a 1.5-fold increase of donor cell engraftment. In summary, our findings demonstrate that p38 may serve as a potential druggable target to restore engraftment of HSPCs in the context of X-CGD gene therapy.
先前针对 X 连锁慢性肉芽肿病（X-CGD）的基因治疗试验缺乏经校正的造血干祖细胞（HSPCs）的长期植入。慢性炎症和白细胞介素 1β（IL1B）水平升高可能导致 X-CGD HSPCs 出现异常细胞周期，同时丧失其长期重编程能力。因此，我们进行了靶向 CRISPR-Cas9 基于 sgRNA 的筛选，以鉴定在基因治疗过程中对抗 HSPC 重编程能力下降的候选基因。在竞争移植测定中对候选基因进行验证，并在使用 IL1B 挑战或 X-CGD HSPC 的疾病背景下进行测试。sgRNA 筛选将 () 鉴定为增加 HSPC 植入的潜在靶标。在移植前敲除 足以诱导选择性优势。抑制 p38 增加了造血干细胞归巢因子 CXCR4 的表达，并减少了 HSPC 中的细胞凋亡和增殖。为了潜在的临床转化，用 p38 抑制剂处理 IL1B 挑战或 X-CGD HSPCs 可使供体细胞植入增加 1.5 倍。总之，我们的研究结果表明，p38 可作为一种潜在的可药物治疗靶点，以恢复 X-CGD 基因治疗中 HSPC 的植入。
