Optimized cell culture conditions promote ex-vivo manipulation and expansion of primitive hematopoietic stem cells for therapeutic gene editing
Optimized cell culture conditions promote ex-vivo manipulation and expansion of primitive hematopoietic stem cells for therapeutic gene editing
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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
DOI：
10.1101/2022.01.11.475795
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Rai R
中科院分区：
文献类型：
--
作者：
Rai R
在过去的几年里，基因编辑已经成为单基因疾病治疗纠正的有力工具。CRISPR/CAS9应用于造血干细胞和祖细胞(HSPC)在治疗血液病的原则验证临床前研究中显示出巨大的前景，使用这些工具的临床试验目前正在进行中。尽管如此，仍然有重要的挑战需要解决，例如靶向原始的、长期重新填充的HSPC的效率以及临床用途的体外扩增。在此，我们测试了不同的培养基组分对HSPC增殖能力的影响，并对报告基因进行同源定向修复，同时在体外培养过程中保留了它们的茎特征。我们测试了不同的化合物组合，并证明通过在培养液中添加组蛋白脱乙酰酶抑制剂，和/或通过微调其细胞因子组成，有可能在体外和体内长期重新填充HSPC中实现高水平的基因靶向，并在保持干性和细胞扩张之间取得有益的平衡，从而与既定的、最先进的培养条件相比，允许获得大量经过编辑的原始HSPC。总体而言，这一优化的体外HSPC培养方案的植入将提高基因编辑的有效性、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。
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 themin vitrofor 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 duringex-vivoculture. 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 bothin vitroandin 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.
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影响因子：
8.8
作者：
Kuo CY;Long JD;Campo-Fernandez B;de Oliveira S;Cooper AR;Romero Z;Hoban MD;Joglekar AV;Lill GR;Kaufman ML;Fitz-Gibbon S;Wang X;Hollis RP;Kohn DB
通讯作者：
Kohn DB
影响因子：
46.9
作者：
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Ploegh HL
影响因子：
17.1
作者：
Schiroli, Giulia;Ferrari, Samuele;Naldini, Luigi
通讯作者：
Naldini, Luigi
影响因子：
8.8
作者：
Shin, Jiyung J.;Schroeder, Markus S.;Corn, Jacob E.
通讯作者：
Corn, Jacob E.
影响因子：
82.9
作者：
Scala, Serena;Basso-Ricci, Luca;Biasco, Luca
通讯作者：
Biasco, Luca