CRISPR/Cas9 Genome-Edited Autologous Hematopoietic Stem Cell Transplantation Therapies for Sickle Cell Disease

Abstract

Hematopoietic stem cell transplantation is the only curative option for sickle cell disease (SCD), a life-threatening genetic disorder that impacts millions worldwide, yet the majority of patients do not have a suitable donor for an allogeneic transplantation. Increased levels of fetal hemoglobin (HbF) are protective against complications of SCD. BCL11A is a transcription factor that binds to the y-globin promoter and controls fetal-to-adult hemoglobin switching. Reducing BCL11A expression or inhibiting its activity can induce expression of HbF in adult red blood cells. We have developed 2 genome editing strategies targeting BCL11A for the induction of HbF levels and report the preclinical development of two potentially curative CRISPR/Cas9 ex-vivo genome-edited autologous hematopoietic stem cell therapies: HIX763 and OTQ923. HIX763 is an investigational cell therapy product harboring a genome modification in the erythroid-specific enhancer region of the BCL11A gene on chromosome 2 resulting in reduced BCL11A expression only in erythroid cells. OTQ923 is an investigational cell therapy product generated by targeted disruption of the γ-globin gene promoter on chromosome 11 to presumably delete BCL11A binding regions in the targeted region. Both HIX763 and OTQ923 are intended to durably increase HbF to therapeutically effective levels to ameliorate complications of SCD. Both products showed editing-dependent upregulation of γ-globin, HbF expression and number of F-cells upon erythroid differentiation of edited hematopoietic stem cells from healthy donors and SCD patients. No off-target editing was identified for both HIX763 and OTQ923 through a complementary suite of genome-wide biochemical and cell-based assays including in silico off-target prediction, SITE-Seq, PanHeme analysis of known oncogenes and tumor suppressors, karyotyping, UnIT evaluation of chromosomal rearrangements, and on-target long-range sequencing analysis. A six-month tumorigenicity study revealed no tumor formation or clonal outgrowth upon transplantation of full human doses of HIX763 and OTQ923 into immunocompromised NOD scid gamma mouse (NSG) mice. Finally, a comparability assessment of OTQ923 produced using the research process and the clinical manufacturing process showed comparability in the quality of cells, as measured by off-target editing, genomic rearrangement, cell viability, editing efficiency, HbF induction, and in vivo engraftment. Together these studies demonstrate that the preclinical data adequately reflects the behavior of the cells intended for clinical use and supports advancing these drug products into clinical trials for the treatment of sickle cell disease.