Conti Anastasia, Giannetti Kety, Midena Federico, Beretta Stefano, Gualandi Nicolò, De Marco Rosaria, Carsana Edoardo, Varesi Angelica, Tavella Teresa, Alessandrini Laura, Zarghamian Parinaz, Weber Alessandra, Ferrari Samuele, Brombin Chiara, Gilioli Diego, Della Volpe Lucrezia, Xie Stephanie Z, Merelli Ivan, Cathomen Toni, Naldini Luigi, Di Micco Raffaella
San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.
San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy; Vita-Salute San Raffaele University, 20132 Milan, Italy.
Cell Rep Med. 2025 Jun 17;6(6):102157. doi: 10.1016/j.xcrm.2025.102157. Epub 2025 Jun 3.
Gene editing (GE) using homology-directed repair (HDR) in hematopoietic stem and progenitor cells (HSPCs) offers promise for long-range gene correction of inherited genetic disorders. However, cellular responses induced by CRISPR-Cas9/AAV6 engineering impair the long-term repopulating potential of HDR-edited HSPCs, adversely impacting the safety and efficacy of clinical translation. Our study uncovers a durable senescence-like response in genetically engineered HSPCs triggered by p53 and interleukin (IL)-1/nuclear factor κB (NF-κB) activation, which restricts graft size and clonal diversity in long-term transplantation assays. We show that transient p53 inhibition or blocking inflammatory pathways mitigates senescence-associated responses, improving the repopulating capacity of edited HSPCs. Importantly, we identify treatment with Anakinra, an IL-1 signaling antagonist, as a promising strategy to enhance polyclonal output in HDR-edited cells while minimizing genotoxicity risks associated with the editing procedure. Overall, our findings present strategies to overcome key hurdles in HDR-based HSPC gene therapies, providing a framework for enhancing their efficacy and safety in clinical applications.
在造血干细胞和祖细胞（HSPCs）中利用同源定向修复（HDR）进行基因编辑（GE）为遗传性遗传疾病的长期基因校正带来了希望。然而，CRISPR-Cas9/AAV6工程诱导的细胞反应损害了HDR编辑的HSPCs的长期重建潜力，对临床转化的安全性和有效性产生了不利影响。我们的研究发现，由p53和白细胞介素（IL）-1/核因子κB（NF-κB）激活引发的基因工程HSPCs中存在持久的衰老样反应，这在长期移植试验中限制了移植物大小和克隆多样性。我们表明，短暂抑制p53或阻断炎症途径可减轻衰老相关反应，提高编辑后的HSPCs的重建能力。重要的是，我们确定用IL-1信号拮抗剂阿那白滞素进行治疗是一种有前景的策略，可增强HDR编辑细胞中的多克隆输出，同时将与编辑程序相关的基因毒性风险降至最低。总体而言，我们的数据提出了克服基于HDR的HSPC基因治疗关键障碍的策略，为提高其在临床应用中的疗效和安全性提供了框架。
