Preclinical Development of HBB Gene Correction in Autologous Hematopoietic Stem and Progenitor Cells to Treat Severe Sickle Cell Disease

Abstract Sickle Cell Disease (SCD) is an autosomal recessive hemoglobinopathy affecting 70-100,000 people in the United States, and millions of people globally. The disease is caused by an adenine to thymidine single nucleotide variant in codon 6 in the human beta-globin gene (HBB gene) that leads to the substitution of glutamic acid by valine (“E6V”) in the beta-globin protein ultimatelyresulting in the formation of sickle beta-globin protein (HgbS) in red cells. While there are therapies like hydroxyurea and red blood cell transfusions that modify the course of the disease, the only curative treatment is allogeneic hematopoietic stem cell transplantation (allo-HSCT), which are available for only 15-20% of the SCD patients, severely limiting the application of allo-HSCT for patients who need it the most. Hence, there remains a tremendous unmet medical need to develop alternative treatment strategies for patients with SCD. An alternative, potentially curative, treatment strategy is to perform gene-editing by homologous recombination (HR) to directly correct the E6V mutation in the HBB gene in autologous hematopoietic stem and progenitor cells (HSPC) from sickle cell patients and then reinfuse these modified cells post-myeloablation. Here we present our IND-enabling preclinical development for CRISPR-Cas9 and rAAV6-mediated HBB genome editing of the E6V mutation in SCD-derived HSPCs as well as in plerixafor mobilized HSPCs from multiple healthy individuals. We demonstrate 30-75% E6V conversion in edited SCD-HSPCs that when differentiated into reticulocytes in vitro, corresponds in the change from HgbS to HgbA tetramers. HBB edited SCD-HSPCs displayed long-term engraftment in the bone marrow of immunodeficient NSG mice at 16 weeks post-transplant, where we identified that ~50% (range of 30-78%) of human cells were edited at the E6V position. Furthermore, by using a recombinant high fidelity Cas9 (HiFi), we were able to eliminate off-target activity as much as 20 fold in edited SCD-HSPCs while maintaining robust on-target activity. Because GCSF use is contraindicated in SCD patients in view of case reports of mortality and that there are ongoing clinical trials using plerixafor to mobilize HSPCs in SCD patients for gene therapy, we also performed IND-enabling HBB genome editing studies in plerixafor mobilized HSPCs from several healthy individuals. We achieved median gene targeting frequencies of ~25% (range of 17-27%) while maintaining high purity (CD34+ percentage >95%) and viability (>80%) across multiple independent experiments. CRISPR-Cas9 and rAAV6- HBB edited plerixafor-HSPCs displayed high potency as evidenced by multilineage methylcellulose progenitor colony formation and importantly, long-term multilineage B, myeloid, and T/NK cell engraftment in the bone marrow of NSG mice at 16-20 weeks post transplant, where a median of ~5% (range of 1-15%) of human alleles were modified at the E6V position. Moreover, In vitro erythroid differentiated human CD34+ HSPCs from NSG mouse bone marrow displayed editing frequencies of 2-15%. These results suggest long-term maintenance of HBB gene targeting in tri-lineage (myeloid/lymphoid/erythroid) plerixafor mobilized repopulating human hematopoietic stem cells. Further optimization of HSPC culture conditions resulted in 2-3 fold higher gene targeting frequencies (up to 70%) and ongoing NSG transplant studies are underway to determine whether the increases in HR frequencies also occur in long-lived repopulating hematopoietic stem cells. Altogether, these preclinical IND-enabling studies outline the framework for the manufacturing of an investigational cellular product consisting of autologous CD34+ HSPC that have been targeted for HBB gene correction of the SCD-causing E6V point mutation for the treatment of severe SCD. Disclosures Vakulskas: IDT: Employment. Behlke: IDT: Employment. Porteus: CRISPRTx: Equity Ownership.