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124 Granulocyte Colony-Stimulating Factor Impairs Engraftment of CRISPR-Cas9 Gene Edited Human Hematopoietic Stem Cells By Exacerbating p53 Mediated DNA Damage Response

Program: Oral and Poster Abstracts
Type: Oral
Session: 801. Gene Therapies: Addressing Challenges & Opportunities in Pre-clinical Settings
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research, hematopoiesis, Biological Processes, molecular biology
Saturday, December 10, 2022: 10:15 AM

Daisuke Araki, MD1, Vicky Chen2*, Neelam Redekar3*, Poching Li4*, Yan Luo4*, Yuesheng Li4* and Andre Larochelle, MD, PhD1

1Laboratory of Regenerative Therapies for Inherited Blood Disorders, Cellular and Molecular Therapeutics Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD
2Integrated Data Science Services, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD
3NIAID Collaborative Bioinformatic Resource (NCBR), NIH, Bethesda, MD
4DNA Sequencing and Genomics Core, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD

Granulocyte colony-stimulating factor (G-CSF) is generally recommended to hasten the recovery of neutropenia after autologous hematopoietic stem cell (HSC) transplantation. However, in patients pre-treated with chemotherapeutic agents, G-CSF was shown to exacerbate HSC toxicity triggered by these drugs by promoting their differentiation or senescence. We reasoned that G-CSF might also negatively impact HSCs treated with DNA-damaging programmable CRISPR/Cas9 nucleases and potentially reduce their engraftment in vivo. To address this question, we previously treated human CD34+ cells by electroporation of AAVS1-specific sgRNA/Cas9 ribonucleoprotein complexes (‘RNP group’) or Cas9 alone (‘Cas9 group’), and transplanted cells from each group into NSG recipient mice. To model conventional autologous transplantation procedures, animals received a daily subcutaneous injection of G-CSF or a control saline (PBS) solution for 14 days post-transplantation. The use of G-CSF post-transplant significantly impaired long-term engraftment of CRISPR/Cas9 gene-edited HSCs (Araki, ASH 2021). In this study, we interrogate at the single-cell level the molecular mechanisms underlying the G-CSF-mediated engraftment defect of gene edited HSCs and provide an approach to overcome the functional impairment.

We analyzed human CD34+ cells harvested from the animals’ bone marrow after G-CSF or PBS treatment using Cellular Indexing of Transcriptomes and Epitopes sequencing (CITE-seq). We identified 7 distinct clusters (clusters 0 to 6) in dimension-reduction analysis. HSCs were computationally assigned to cluster 6 by associating cluster-specific transcripts with HSC signatures and by comparing CITE-seq CD38, CD45RA, CD90, CD49f expression. Gene set enrichment analysis (GSEA) of cells within the HSC cluster revealed a significant upregulation of interferon α/γ response pathways in G-CSF treated HSCs in both RNP and Cas9 groups, indicating that G-CSF stimulated an inflammatory response in engrafted human HSCs in-vivo independent of the gene editing process (Fig A). In contrast, significant G-CSF-mediated up-regulations of the p53 pathway, a central network orchestrating DNA-damage response (DDR) mechanisms, and of the cellular apoptotic response were observed only in gene edited HSCs (Fig A). In addition, the G2-M DNA damage checkpoint, a key cell cycle regulator that prevents entry into mitosis until damaged DNA is sufficiently repaired, was exclusively downregulated in the RNP group by G-CSF treatment (Fig. A). To further substantiate the impact of G-CSF on gene edited HSCs, we transiently inhibited the p53 pathway by co-electroporating GSE56 mRNA during gene editing (‘RNP/GSE56 group’), the approach previously shown to improve graft function of gene edited HSCs (Schiroli, Cell Stem Cell 2019). Addition of GSE56 mRNA offset the transcriptional differences previously observed between G-CSF treated and untreated gene edited HSCs. Next, to evaluate whether the transcriptional changes induced by GSE56 altered engraftment of gene edited LT-HSCs in vivo, we transplanted CD34+ cells electroporated with RNP/GSE56 into NSG mice and injected G-CSF or PBS once daily for the first 14 days after cell infusion. Notably, we observed a comparable long-term (22 weeks) human cell engraftment between G-CSF treated and untreated mice, suggesting that transient p53 inhibition ameliorated the negative effects of G-CSF on gene edited LT-HSC function.

In conclusion, our data indicate that G-CSF administration post-transplant impairs long-term engraftment of CRISPR/Cas9 gene-edited HSCs by exacerbating the p53-mediated DDR triggered by Cas9-mediated double stranded DNA breaks (Fig B). Perturbation of the G2-M DNA damage checkpoint induced by G-CSF may further contribute to the molecular mechanisms underlying the engraftment defect of gene edited HSCs. As clinical trials are implemented, the potential for G-CSF to exacerbate HSC toxicity mediated by DNA-damaging nucleases should be considered.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH