During the last few years, gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR/Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat haematological disorders, and clinical trials using these tools are now underway. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and expand them in vitro for clinical purposes. Here we have tested the effect exerted by different culture media compositions on the ability of HSPCs to proliferate and undergo homology directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during ex-vivo culture. We tested different combinations of compounds and demonstrated that by supplementing the culture media with inhibitors of histone deacetylases, and/or by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both in vitro and in vivo, with a beneficial balance between preservation of stemness and cell expansion, thus allowing to obtain a significant amount of edited, primitive HSPCs compared to established, state-of-the-art culture conditions. Overall, the implantation of this optimized ex vivo HSPC culture protocol will improve the efficacy, feasibility and applicability of gene editing and will likely provide one step further to unlock the full therapeutic potential of such powerful technology.

中文翻译：

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优化的细胞培养条件促进原始造血干细胞的离体操作和扩增，用于治疗性基因编辑

在过去几年中，基因编辑已成为治疗纠正单基因疾病的有力工具。应用于造血干细胞和祖细胞 (HSPC) 的 CRISPR/Cas9 在治疗血液疾病的原理验证临床前研究中显示出巨大的前景，目前正在使用这些工具进行临床试验。尽管如此，仍然存在需要解决的重要挑战，例如靶向原始的、长期重新填充的 HSPC 的效率，并将它们在体外进行扩展以用于临床目的。在这里，我们测试了不同培养基组合物对 HSPC 增殖和经历同源定向修复介导的报告基因敲入能力的影响，同时在离体过程中保留其干性特征文化。我们测试了不同的化合物组合，并证明通过在培养基中添加组蛋白去乙酰化酶抑制剂，和/或通过微调其细胞因子组成，可以在体外和体外长期再生 HSPC 中实现高水平的基因靶向。在体内，在保持干性和细胞扩增之间取得了有益的平衡，因此与已建立的最先进的培养条件相比，可以获得大量经过编辑的原始 HSPC。总体而言，这种优化的体外 HSPC 培养方案的植入将提高基因编辑的功效、可行性和适用性，并可能进一步释放这种强大技术的全部治疗潜力。