Xu Liwen, Lahiri Premanjali, Skowronski Jason, Bhatia Neehar, Lattanzi Annalisa, Porteus Matthew H
Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
Stanford Laboratory for Cell and Gene Medicine, Stanford University, Stanford, CA 94304, USA.
Mol Ther Methods Clin Dev. 2023 Jul 27;30:317-331. doi: 10.1016/j.omtm.2023.07.009. eCollection 2023 Sep 14.
gene correction with CRISPR-Cas9 and a recombinant adeno-associated virus serotype 6 (rAAV6) in autologous hematopoietic stem/progenitor cells (HSPCs) to treat sickle cell disease (SCD) has now entered early-phase clinical investigation. To facilitate the progress of CRISPR-Cas9/rAAV6 genome editing technology, we analyzed the molecular changes in key reagents and cellular responses during and after the genome editing procedure in human HSPCs. We demonstrated the high stability of rAAV6 to serve as the donor DNA template. We assessed the benefit of longer HSPC pre-stimulation in terms of increased numbers of edited cells. We observed that the p53 pathway was transiently activated, peaking at 6 h, and resolved over time. Notably, we revealed a strong correlation between p21 mRNA level and rAAV6 genome number in cells and beneficial effects of transient inhibition of p53 with siRNA on genome editing, cell proliferation, and cell survival. In terms of potential immunogenicity, we found that rAAV6 capsid protein was not detectable, while a trace amount of residual Cas9 protein was still detected at 48 h post-genome editing. We believe this information will provide important insights for future improvements of gene correction protocols in HSPCs.
利用CRISPR-Cas9和重组腺相关病毒6型（rAAV6）对自体造血干/祖细胞（HSPC）进行基因校正以治疗镰状细胞病（SCD）现已进入早期临床研究阶段。为推动CRISPR-Cas9/rAAV6基因组编辑技术的发展，我们分析了人类HSPC基因组编辑过程中及之后关键试剂的分子变化和细胞反应。我们证明了rAAV6作为供体DNA模板具有高度稳定性。我们评估了延长HSPC预刺激时间对增加编辑细胞数量的益处。我们观察到p53通路被短暂激活，在6小时达到峰值，并随时间逐渐消退。值得注意的是，我们发现细胞中p21 mRNA水平与rAAV6基因组数量之间存在强相关性，以及用小干扰RNA（siRNA）短暂抑制p53对基因组编辑、细胞增殖和细胞存活具有有益影响。在潜在免疫原性方面，我们发现无法检测到rAAV6衣壳蛋白，而在基因组编辑后48小时仍能检测到微量残留的Cas9蛋白。我们相信这些信息将为未来改进HSPC基因校正方案提供重要见解。
Zotero 插件
