ENAapplication/xmlftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/087/SRR26204587/SRR26204587_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/052/SRR33439752/SRR33439752_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/079/SRR26204579/SRR26204579_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/048/SRR33439748/SRR33439748_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/047/SRR33439747/SRR33439747_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/086/SRR26204586/SRR26204586_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/050/SRR33439750/SRR33439750_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/078/SRR26204578/SRR26204578_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/077/SRR26204577/SRR26204577_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/081/SRR26204581/SRR26204581_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/051/SRR33439751/SRR33439751_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/088/SRR26204588/SRR26204588_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/078/SRR26204578/SRR26204578_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/082/SRR26204582/SRR26204582_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/080/SRR26204580/SRR26204580_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/084/SRR26204584/SRR26204584_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/050/SRR33439750/SRR33439750_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/085/SRR26204585/SRR26204585_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/081/SRR26204581/SRR26204581_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/083/SRR26204583/SRR26204583_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/087/SRR26204587/SRR26204587_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/052/SRR33439752/SRR33439752_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/084/SRR26204584/SRR26204584_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/049/SRR33439749/SRR33439749_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/047/SRR33439747/SRR33439747_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/086/SRR26204586/SRR26204586_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/049/SRR33439749/SRR33439749_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/051/SRR33439751/SRR33439751_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/079/SRR26204579/SRR26204579_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/085/SRR26204585/SRR26204585_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/088/SRR26204588/SRR26204588_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR334/048/SRR33439748/SRR33439748_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/080/SRR26204580/SRR26204580_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/082/SRR26204582/SRR26204582_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/083/SRR26204583/SRR26204583_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR262/077/SRR26204577/SRR26204577_2.fastq.gzprimaryOK200GenomicsSan Raffaele Telethon Institute for Gene Therapyhttps://www.ebi.ac.uk/ena/browser/view/PRJNA1022002Homo 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 24 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]2025-09-242025-03-22PRJNA1022002GSE244247960640466639