ENAapplication/xmlftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/028/SRR26205228/SRR26205228.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/031/SRR26205231/SRR26205231.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/082/SRR33434682/SRR33434682.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/025/SRR26205225/SRR26205225.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/081/SRR33434681/SRR33434681.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/079/SRR33434679/SRR33434679.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/032/SRR26205232/SRR26205232.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/021/SRR26205221/SRR26205221.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/023/SRR26205223/SRR26205223.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/024/SRR26205224/SRR26205224.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/080/SRR33434680/SRR33434680.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/027/SRR26205227/SRR26205227.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/077/SRR33434677/SRR33434677.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/030/SRR26205230/SRR26205230.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/026/SRR26205226/SRR26205226.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/078/SRR33434678/SRR33434678.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/029/SRR26205229/SRR26205229.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/022/SRR26205222/SRR26205222.fastq.gzprimaryOK200GenomicsSan Raffaele Telethon Institute for Gene Therapyhttps://www.ebi.ac.uk/ena/browser/view/PRJNA1022001Homo sapiensxref:PubMed:40466639Gene 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, adverse cellular responses induced by CRISPR-Cas9/AAV6 engineering impair the long-term repopulating potential of HDR-edited HSPCs, limiting clinical translation. Our study uncovers a senescence-like response in genetically-engineered HSPCs triggered by p53 and IL-1/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 can mitigate senescence-associated responses, enhancing the repopulating capacity of edited HSPCs. Importantly, we identify Anakinra, an IL-1 signaling antagonist, as a safer and effective strategy to enhance polyclonal output in HDR-edited cells while minimizing genotoxicity risks associated with the gene-editing procedure. These findings present strategies to overcome key hurdles in HDR-based HSPC therapies, providing a framework for enhancing the efficacy and safety of these approaches in future clinical applications. Overall design: Cord-blood (CB) hematopoietic stem and progenitor cells (HSPCs) were cultured for three days in early active and stem-preserving medium and at day 3 we performed the following treatments: the electroporation with an RNP with no predictive activity in the human genome in presence (RNP NEG + ANAK) or absence (RNP NEG) of Anakinra and the standard gene editing procedure with HS RNP and transduction with an AAV6 carrying the DNA donor template for HDR in presence (HS/AAV6 + ANAK) or absence (HS/AAV6) of Anakinra. After 96 hours post treatments, HSPCs were collected for RNA-seq analysis.ENAfalseSenescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6 mediated gene editing in hematopoietic stem cells [RNA-seq]Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6 mediated gene editing in hematopoietic stem cells [RNA-seq]2025-09-242025-03-22PRJNA1022001GSE244248960640466639