Development of a β-Globin Gene Replacement Strategy As a β-Thalassemia Therapy

Abstract

Background: β-thalassemia is a genetic disorder with high unmet need worldwide. Over 300 genetic mutations that result in the reduction, or loss, of β-globin (HBB) expression have been characterized to date. Patients with thalassemic mutations in both HBB alleles often experience severe anemia resulting from ineffective erythropoiesis due to the loss of adult hemoglobin (HbA) tetramers (α2β2) and imbalanced excess of α-globin chains. One challenge is creating a genome-editing strategy that replaces the diversity of disease-causing mutations in the HBB gene while restoring physiological HbA expression. Therefore, a gene-editing platform using homology-directed repair (HDR) that could replace the entire HBB gene in situ and under the control of normal regulatory elements would be an ideal strategy. Methods: CD34+ hematopoietic stem and progenitor cells (HSPCs) were edited using high-fidelity Cas9, HBB guide RNA, and different AAV6 DNA donor templates and then differentiated into red blood cells in vitro. We evaluated HBB expression in 39 AAV6 donors engineered with heterologous introns and different polyadenylation signals first using a flow cytometry screening assay and later HPLC. Candidates that met or exceeded wild-type HBB expression were moved forward for further comparison. Next, we edited HSPCs with the 8 highest expressing AAV6 donors, performed in vitro differentiation, and evaluated HDR frequencies by droplet digital polymerase chain reaction (ddPCR) and HbA formation by HPLC. The candidate that demonstrated the highest conversion of donor knock-in to HbA was further engineered to boost expression and reduce construct size. Two final candidates were moved forward to further characterize their ability to rescue loss of HbA in CD34+ HSPCs isolated from patients with sickle cell disease (SCD) at the protein and RNA level. Additionally, we performed DNA analysis on the gene-edited cells using ddPCR and a targeted PCR-free long-range sequencing approach. Results: Adding heterologous introns to the HBB coding sequence significantly increased HBB expression relative to the no-intron construct containing only coding DNA. The results from our flow cytometry screen and HPLC analysis were positively correlated, and the top DNA donors with heterologous introns resulted in physiologic HbA expression. Using this gene-replacement strategy, we achieved HDR frequencies of up to 40% in CD34+ HSPCs. Further optimization resulted in a smaller DNA template, maintenance of HbA conversion rates, and a lower rate of unintended recombinations. Finally, using HSPCs from patients with SCD as a therapeutically relevant model, we found, by HPLC and RNA sequencing, the optimized DNA donor can successfully replace the entire nonfunctional HBB gene, thereby restoring physiologic HbA expression. Future experiments are investigating in vitro gene correction in cells isolated from patients with β-thalassemia and additional studies evaluating in vivo stem cell repopulation capacity of gene-edited HSPCs. Conclusion: We developed an HBB AAV6 donor engineered with heterologous introns that demonstrates efficient HBB gene replacement and HbA rescue in the erythroid progeny of CD34+ HSPCs isolated from patients with SCD, offering a potential differentiated approach for treating β-thalassemia.