Cellular senescence and inflammatory programs are unintended consequences of CRISPR-Cas9 gene editing in hematopoietic stem and progenitors cells

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: Gene editing (GE) in hematopoietic stem and progenitor cells (HSPCs) is a revolutionary site-specific gene correction strategy for a plethora of immune-hematological diseases. Despite the rapid development of advanced GE-based therapies, a few challenges remain to be faced to improve GE efficiency and the reduced repopulating potential upon transplantation. We previously showed that the combination of nuclease-induced Double Strand Break with DNA repair template for Homology Directed Repair (HDR) delivered by AAV6 caused cumulative activation of the p53-mediated DNA Damage Response (DDR) pathway constraining HSPC proliferation and yield, suggesting that DDR-related cellular programs may inadvertently contribute to HSPC dysfunction upon GE. Protracted DDR signaling has been linked to the establishment of cellular senescence, a condition in which cells, despite being still alive, are unable to further proliferate and are characterized by activation of inflammatory programs. Yet, whether GE could have durable and long-term consequences on the functionality of HSPCs remains to be elucidated. Aims: The overreaching goals of the project are to elucidate the long-term impact of the GE procedure on HSPCs and to develop new strategies to counteract edited HSPC dysfunction for more efficient gene therapies. Methods: GE was performed in human cord blood (CB) or mobilized peripheral blood (mPB)-derived HSPCs with an AAV6 carrying a HDR donor template with a GFP reporter gene to enable easy detection of edited cells. To follow HSPC repopulating potential long-term, cells were injected into sub-lethally irradiated NOD Prkdcscid Il2rg−/− mice and human cell engraftment was monitored in peripheral blood and in the hematopoietic organs by flow cytometry. We evaluated senescence activation upon GE by flow cytometry for senescence markers as well as by ATAC-seq and RNA-seq for inflammatory and senescence-associated genes. We performed the clonal tracking of individual HDR-edited HSPCs thanks to our barcoding-based strategy. Results: By integrating transcriptional analysis (up to the single cell level) with innovative imaging-based cellular assays we reported induction of cellular senescence markers (p16 and Senescence-Associated β-Galactosidase) and pro-inflammatory programs across edited HSPC subtypes and in vivo in the human graft. Consistently, we found open chromatin at promoters of several senescence-gene categories and inflammatory genes of the IL1 axis (an upstream mediator of DDR-dependent inflammation) and NF-kB pathway (a key regulator of inflammatory genes) especially in HDR-edited cells. Transcriptional activation of inflammatory cytokines in edited HSPCs was DDR-dependent and partly mitigated by transient p53 inhibition. In this context, temporary inhibition of IL1 and NF-kB pathways at the time of GE resulted in increased clonogenicity of edited HSPCs ex vivo and ameliorated long-term hematopoietic reconstitution in xenotranspanted mice. We also reported a decrease in senescence markers in both CB and mPB-derived HSPCs upon GE. In vivo clonal tracking of HDR-edited HSPCs revealed that IL1 inhibition improved polyclonal reconstitution and better preserved self-renewal and multi-potency of individual edited HSPCs. Summary/Conclusion: Our findings define senescence and inflammatory programs as long-term consequences of CRISPR-Cas9 engineered human HSPCs and pave the way for the development of novel strategies based on senescence modulation and anti-inflammatory molecules to overcome adverse cellular responses for efficient HSPC-based clinical application. Keywords: Adeno-associated virus, IL-1, Gene therapy, Hematopoietic stem cell