Genome Editing Recreates Hereditary Persistence of Fetal Hemoglobin in Primary Human Erythroblasts

Manipulating the developmental switch from γ- to β-globin expression that occurs after birth has been intensively investigated as therapeutic strategy for sickle cell anemia and β-thalassemia. Rare individuals with a benign condition termed hereditary persistence of fetal hemoglobin (HPFH) exhibit an attenuated or absent γ-to-β switch, resulting in high levels of fetal hemoglobin (α2γ2) in all red blood cells (RBCs) throughout life. Moreover, individuals with HPFH and homozygosity for sickle cell disease (SCD) mutations exhibit few or no clinical manifestations of the latter. We used genome editing to induce a naturally occurring 13-nucleotide (-102 to -114) deletional HPFH mutation in the γ-globin ( HBG1 ) gene promoter. Heterozygosity for this mutation is associated with HbF levels > 30% in adults. We used the clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system to create small deletions around -102 to -114 in the γ-globin genes in peripheral blood CD34+ cells from healthy donors. We delivered guide RNA (gRNA) and Cas9 using lentiviruses, sorted transduced hematopoietic progenitors by FACS, and cultured them using a 3-phase erythroid differentiation protocol. Real time PCR showed that γ-globin mRNA increased more than 10-fold in Cas9/gRNA transduced cells compared to controls. HbF flow cytometry and high-performance liquid chromatography (HPLC) demonstrated that induced γ-globin chains were effectively incorporated into hemoglobin tetramers. HPLC revealed 1-3% HbF in negative controls and an increase to 15% in cells transduced with gRNA and Cas9. Expression of erythroid differentiation markers CD235 and CD71 were unaffected, suggesting that the γ-globin increase is not due to impaired erythroid maturation. Next generation sequencing demonstrated that a single gRNA created one predominant mutation that co-segregated with high HbF expression and represented over 50% of the sequencing coverage. Interestingly, this mutation is identical to the 13-nucleotide HPFH deletion. We also tested the gRNA mutation efficiency after transient expression of gRNA and Cas9 in human CD34+ cells by electroporation followed by analysis of single burst-forming unit-erythroid (BFU-E) colonies formed in methylcellulose. Genomic DNA analysis revealed that one gRNA targeted 50% of HBG1 alleles, and cells that received two overlapping gRNAs demonstrated 80% mutation frequency. Real-time PCR of mRNA from edited BFU-Es showed that mutations stimulated γ-globin mRNA expression to 19-55% total globin synthesis, whereas control colonies contained 1-5% γ-globin. Together, our data demonstrate that the CRISPR-Cas9 system can generate precisely the -102 to -114 HPFH mutation at high efficiency in primary human progenitor cells and thereby induce the expression of HbF to potentially therapeutic levels. This work provides proof of concept for targeted genome editing for γ-globin activation as a therapy for patients with β hemoglobinopathies. Disclosures Weiss: Biogen: Research Funding; GlaxoSmithKline: Consultancy; Rubius: Membership on an entity9s Board of Directors or advisory committees.