Α-Globin Lentiviral Vectors for Hematopoietic Stem Cell Gene Therapy of α-Thalassemia

α-thalassemia is an inherited blood disorder caused by mutations in α-globin genes (HBA1 and HBA2) resulting in the reduction of α-globin chains, the subunit, along with β-globin chain, constituting adult hemoglobin (α ₂β ₂). While a reduction of α-globin expression of 40% presents asymptomatic phenotypes, expression levels decreasing to <20% or an α/β-globin mRNA ratio of <0.2, causes severe anima. Treatment for severe cases of α-thalassemia entails lifelong, biweekly blood transfusions with daily chelation therapy. While these therapies enable patients to live into mid- to late-adulthood, they continue to engender serious clinical manifestations, such as iron toxicity.

To answer this clinical need, our laboratory has developed a stem cell gene therapy in which functional copies of α-globin gene integrate into the genome of patient's hematopoietic and progenitor stem cells (HSPCs) by lentiviral vectors (LVs) to normalize the globin chain imbalance and restore hemoglobin function in derived red blood cells (RBCs). The design of our α-globin LVs (AGLVs) is based on GLOBE β-globin LV utilized in a clinical trial for β-thalassemia (NCT02453477), which has achieved transfusion reduction and independence in patients with transfusion-dependence β-thalassemia major. We have constructed a series of short proviral length AGLVs for optimized titer production, HSPC infectivity, and lineage-specific α-globin gene expression.

Twelve AGLVs varying in gene and regulatory element compositions were constructed and assessed for raw titer yields and characterized for gene transfer efficiency, mRNA expression and hemoglobin production in a α-globin knockout (KO) human erythroid cell line. Successfully, all AGLVs confer high raw titers ~ 1e7 TU/mL and enabled the production of adult hemoglobin (HbA). We identified two optimal AGLVs for further characterization in human primary HSPCs: 1) A2-UV for yielding highest raw titers and gene transfer efficiency, and 2) A2-Globe for producing the most α-globin mRNA. Both A2-UV and A2-Globe LVsharbor HBA2, are regulated by the β-globin promoter and enhanced by the core or large β-LCR enhancer region, respectively. To assess candidate AGLVs in human HSPCs, AGLVs were tagged at the transcription level to enable identification and quantification of vector-derived α-globin mRNA proceeding HSPCs transduction anderythroid differentiation. A2-UV demonstrated optimum CD34 ⁺ infectivity and gene transfer, and A2-Globe expressed a high ~30% of α-globin mRNA per total β-globin per vector copy number (VCN), achieving levels of one endogenous α-globin gene (~25% per total β-globin).

Current studies examined A2-UV and A2-Globe in α-thalassemia major patient HSPCs - lacking all four α-globin genes - harboring a lethal homozygous 20kb deletion of HBA1 and HBA2, known as SEA ^–/– mutation. Patient HSPCs were transduced and differentiated into RBCs. A high CD34+ infectivity and gene transfer was obtained by both candidate vectors, without utilization of transduction enhancers. Furthermore, a correction of α/β-globin mRNA ratio of ~0.7 was obtained while retaining a low VCN (<3).RBCs were then analyzed at the protein level . Chromatograms of hemoglobin tetramers revealed that HbA represented 98% of all hemoglobin, compared to 0% in non-transduced patient RBCs. Single globin chain chromatograms successfully demonstrated α/β-globin chain ratio of ~0.4 or higher and successfully demonstrated that a low average of copies per cell (~3) restored 60% of adult hemoglobin when normalized to healthy donor RBCs. These data demonstrate α/β-globin correction and hemoglobin restoration in α-thalassemia patient cells with the most severe mutation. Three assays were conducted on patient cells, all with similar and highly encouraging results for disease amelioration in severe cases of α-thalassemia. Overall, this project is paving the way for the first development of a curative gene therapy approach for α-thalassemia.