An Integrated Gene Therapy Strategy for γ-Globin Addition and HbF Reactivation in β-Thalassemia

Abstract

Background: β-Thalassemia and other β-hemoglobinopathies arise from defective β-globin production, and reactivating fetal hemoglobin (HbF) is a well-established strategy to ameliorate disease severity. Two principal gene-therapy approaches—erythroid-specific γ-globin gene addition and shRNA/shmiRNA-mediated knockdown of the γ-globin repressor BCL11A—have each shown clinical promise, but their combination within a single vector remains largely unexplored. Methods: Here we developed and compared three compact, C1-insulated lentiviral vectors driven by the micro-locus control region (μLCR): a BCL11A-targeting shmiRNA vector, a γ-globin cDNA vector, and a dual construct combining both elements. Results: All vectors were produced at high titers, and incorporating both cassettes into a single construct did not compromise vector production or cell viability. In CD34+ hematopoietic stem and progenitor cells from healthy donors and β-thalassemia patients, transduction did not impair proliferation, erythroid differentiation, or progenitor colony formation. All three vectors significantly increased the proportion of HbF-expressing cells, with the combined shmiRNA + γ-globin cDNA vector achieving the highest induction, reaching approximately 25% HbF-positive cells in the enucleated erythroid population of thalassemic cells versus approximately 10% in untransduced controls. Notably, all constructs significantly reduced reactive oxygen species levels, indicating alleviation of oxidative stress. Conclusions: Together, these findings support the feasibility and therapeutic potential of combining complementary HbF-inducing mechanisms within a single lentiviral vector for β-thalassemia.