Deciphering unintended large gene modifications in gene editing for sickle cell disease

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Summary: Sickle cell disease (SCD) is a genetic disease that affects millions of people worldwide, with significant morbidity and a median life expectancy in the mid-forties. Although SCD can be cured by allogeneic hematopoietic stem cell transplantation (alloHSCT), this treatment strategy has substantial limitations and is only available to ~15% of patients. CRISPR/Cas9 based genome-editing strategies for treating SCD have been developed by either correcting the sickle mutation in β-globin (HBB) gene or disrupting the BCL11A erythroid enhancer in patients’ hematopoietic stem and progenitor cells (HSPCs). Multiple clinical trials using gene editing strategies have received FDA approval, and the Phase 1 clinical trial (NCT03745287) by Vertex Pharmaceuticals and CRISPR Therapeutics has shown promise. We have discovered recently that CRISPR/Cas9 genome editing can induce unintended large gene modifications, such as large deletions, insertions and complex local rearrangements, at the Cas9 on-target cut-site. Our results show that large deletions of up to several thousand bases occurred with high frequencies at/near the Cas9 on-target cut-sites on the HBB (11.7-35.4%), HBG (14.3%), and BCL11A (13.2%) genes respectively in HSPCs from patients with SCD. However, the persistence and biological consequences of these large gene modifications are largely unknown, the mechanisms of generating large deletions and insertions remain elusive, and no method is available to reduce the unwanted large gene modifications. There is an unmet need to determine the clinical implications of the unintended large gene modifications in gene-edited SCD HSPCs. The central hypothesis of the proposed research is that a good understanding of the persistence and functional consequences of unintended large gene modifications and the ability to control them will increase the efficacy and safety of gene-editing based treatment of SCD. In Aim 1 studies we will determine the ineffective maturation and HbF induction due to large gene modification in gene edited SCD HSPCs by performing SMRT-seq and single-cell RNA analysis. In Aim 2 we will determine the persistence of large gene modifications in HBB and BCL11A alleles after engraftment of gene-edited SCD HSPCs into mice and patients undergoing CRISPR/Cas9 gene- editing based SCD clinical trials. In Aim 3 we will develop strategies to minimize the detrimental large deletions by establishing a better understanding of the competition between different DNA damage repair pathways and designing and optimizing ssODN templates and short gRNAs as blockers. These studies will address an unmet need in the therapeutic genome editing field and facilitate the translation of genome editing based SCD treatment into clinical practice.