S. N. Chu1,2,5, E. Soupene3, B. Wienert4, H. Yin5, D. Sharma1,2, K. Jia1,2, S. Homma6, J. P. Hampton7, B. R. Conklin4, T. C. MacKenzie1,2, M. Porteus7, M. K. Cromer1,2,8 1University of California, San Francisco, Department Of Surgery, San Francisco, CA, USA 2University of California, San Francisco, Eli & Edythe Broad Center For Regeneration Medicine, San Francisco, CA, USA 3University of California, San Francisco, Children’s Hospital Of Oakland Research Institute, Oakland, CA, USA 4University of California, San Francisco, Department Of Medicine, San Francisco, CA, USA 5University of California, San Francisco, Diabetes Center, San Francisco, CA, USA 6Stanford University, Department Of Genetics, Stanford, CA, USA 7Stanford University, Department Of Pediatrics, Stanford, CA, USA 8University of California, San Francisco, Department Of Bioengineering & Therapeutic Sciences, San Francisco, CA, USA
Introduction: α-thalassemia major (ATM) is an autosomal recessive disorder where all four copies of the α-globin gene are deleted. Allogeneic hematopoietic stem cell transplant (HSCT) has a limited role in the treatment of ATM, due to the paucity of suitable donors and high risk of morbidity and mortality due to prerequisite myeloablative regimens. Here, we describe a CRISPR/AAV-mediated genome editing strategy to restore a full-length copy of the α-globin gene at the β-globin locus in ATM patient-derived HSCs to restore normal hemoglobin production to a patient’s red blood cells (RBCs).
Methods: We identified a Cas9 gRNA that efficiently introduced indels at the HBB locus as well as an AAV6 vector that mediated efficient integration into the start codon of HBB. After designing and testing integration cassettes, we developed two candidate vectors that insert a HBA1 transgene at the HBB locus, with one bicistronic vector that also expresses a truncated form of the erythropoietin receptor (tEPOR) to increase erythropoietic output from edited HSCs. CD34+ HSCs from three patients with ATM were edited and cultured for 14 days in RBC differentiation media. Editing rates were quantified by ddPCR and bulk RNA sequencing was performed. Lastly, α-globin protein production was quantified using high protein liquid chromatography (HPLC).
Results: Edited cells with the combined HBA+tEPOR vector demonstrated superior editing frequencies and more than double total cell counts by the end of RBC differentiation. RNA sequencing of edited ATM-derived HSCs revealed a restoration of HBA1 and HBA2 gene expression. These results were supported by HPLC, which demonstrated production of normal hemoglobin tetramers (HbF, HbA, HbA2) compared to unedited patient cells [Figure 1]. Despite integration at the HBB locus, neither RNA sequencing nor single globin HPLC showed disruption of β-globin production.
Conclusion: To our knowledge, these results demonstrate for the first time that genome editing may be used to increase α-globin production in ATM patient-derived RBCs. We found that the editing strategy that paired site-specific α-globin integration with tEPOR expression achieved the highest editing frequencies, greatest production of edited cells, and the most robust production of α-globin protein compared to α-globin integration alone. This approach has the potential to overcome some of the clinical challenges seen in HSCT for the hemoglobinopathies, including low editing and engraftment rates, as well as potentially reduce or eliminate the need for myeloablation. These findings support development of a definitive ex vivo autologous genome editing strategy that may be curative for ATM.
