Crispr/Cas9- Mediated Genome Editing of Human CD34+ Cells Upregulate Fetal Hemoglobin to Clinically Relevant Levels in Single Cell-Derived Erythroid Colonies

Fetal hemoglobin (HbF) is abundant in the late stage fetus and newborns, but is progressively lost over the first 6 months of life as the genes encoding the g-globin subunit of HbF are repressed. HbF expression can provide clinical benefit to patients with deficient or defective b-globin, for example in β-thalassemia and Sickle Cell Disease (SCD), respectively. CRISPR-Cas9 technology offers a unique treatment modality that can be used to ex vivo edit regulatory DNA sequences in patient CD34 + hematopoietic stem and progenitor cells (HSPC) containing hematopoietic stem and progenitor cells in order to upregulate HbF. Reinfusion of edited CD34 + HSPC would be expected to lead to the production of erythrocytes expressing high levels of HbF, and amelioration of disease. There are a number of reports of naturally occurring genetic variants that are associated with Hereditary Persistence of Fetal Hemoglobin (HPFH), presumably through the loss of regulatory sequences that would otherwise bind regulatory proteins that developmentally down-regulate the expression of HbF. Thus, by using the CRISPR-Cas9 system to re-create the DNA sequence variations associated with HPFH in CD34 + HSPC, we aim to relieve transcriptional inhibition of ɣ-globin, resulting in upregulation of HbF. Here, we transfected healthy primary human CD34 + HSPC with Cas9 and guide RNAs in order to re-create naturally occurring HPFH genetic variants. Transfected CD34 + HSPC were sorted as single cells post-editing and expanded as erythroid colonies. These colonies were then genotyped to confirm editing and further assessed for globin transcript and protein. Clonal analysis demonstrated Non-Homologous End Joining (NHEJ) mediated editing in greater than 90% of the colonies. These editing events re-created the intended HPFH genetic variants in up to 75% of the colonies. For some, but not all of the HPFH genetic variants, both mono-and bi-allelic genetic modifications led to significant upregulation of HbF. These results provide us with an editing strategy that support a viable therapeutic approach for the treatment of β-thalassemia and SCD. Disclosures Mishra: CRISPR Therapeutics: Employment. Chou: CRISPR Therapeutics: Employment. Lin: CRISPR Therapeutics: Employment. Paik: CRISPR Therapeutics: Employment. Zhang: CRISPR Therapeutics: Employment, Equity Ownership. Liang: CRISPR Therapeutics: Employment. Tomkinson: CRISPR Therapeutics: Employment. Pettiglio: CRISPR Therapeutics: Employment. Sanginario: CRISPR Therapeutics: Employment. Allen: CRISPR Therapeutics: Employment. Cradick: CRISPR Therapeutics: Employment. John: CRISPR Therapeutics: Employment. Chakraborty: CRISPR Therapeutics: Employment. Cowan: CRISPR Therapeutics: Employment, Equity Ownership. Novak: CRISPR Therapeutics: Employment, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees. Lundberg: CRISPR Therapeutics: Employment, Equity Ownership.