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

Biography:

Eva Segura originally from France but have done her schooling in the United States. She graduated from University of California Los Angeles (UCLA) with a Bachelor of Science in Biochemistry in 2018. In her undergraduate studies she worked with Dr. Eric Vilain and Dr. Margot Quinlan in the Human Genetics Department and the Biochemistry Department, respectively. Her experiences working with Drs. Vilain and Quinlan gave her an extensive background in molecular biology and galvanized her to pursue a doctoral degree in translational research. Now she is a 4th year Ph.D. candidate in the Molecular Biology Interdepartmental Program at UCLA, in Dr. Donald Kohn’s laboratory, recognized for developing a gene therapy cure for ADA-SCID. Her doctoral research is to develop a hematopoietic stem cell gene addition therapy for alpha thalassemia, an inherited blood disorder, to improve erythropoiesis and restore hemoglobin function.

Abstract

Background: 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 (α₂β₂). Severe α-thalassemia arises with α-globin expression levels of <30% or less, and α/β-globin ratio of <0.2, caused by defects in or absence of three or all four α-globin genes. Treatment for survival 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.

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 cell (HSPC) by lentiviral vectors (LVs) to normalize the globin chain imbalance and restore hemoglobin function. 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 independence in patients with transfusion-dependence β-thalassemia major. To target excess infective erythropoiesis and hemoglobin restoration, we have constructed a series of short proviral length AGLVs for optimized titer production, HSPC infectivity, and gene expression.
Research: 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 were able to yield adult hemoglobin. We identified two optimal AGLVs for further characterization in human primary HSPCs: 1) Alpha2 LV for yielding highest raw titers and gene transfer efficiency, and 2) LCR-Globe LV for producing the most α-globin mRNA. Both Alpha2 and LCR-Globe LVs harbor 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 and erythroid differentiation. Alpha2 demonstrated optimum CD34⁺ infectivity and gene transfer, and LCR-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).We then assessed one of these vectors in Alpha Thalassemia Major (ATM) patient HPSCs – lacking all four α-globin genes. We successfully demonstrated that a low average of copies per cell (~2) restored adult hemoglobin formation by 50%, which was determined by measuring α-globin chains (produced by the introduced α-globin gene) to other endogenous β-globin chains. (To note, two endogenous α-globin genes, or 50% gene expression, result in asymptomatic cases of α-thalassemia in most patients). We further showed that transduced cells are similar to healthy RBCs; round, pale enucleated RBCs, unlike the untransduced patient RBCs which contained more nucleated cells (sign of ineffective RBC maturation). These preliminary patient cell results are promising and will be repeated with further morphologic and gene expression measurements.Based on these positive results, we are confidently moving forward with animal studies. The selected mouse model contains a mild form of α-thalassemia caused by a deletion of two α-globin genes and present human-like symptoms of AT, such as anemia and an enlarged spleen. These vectors have been converted to murine-carrying α-globin., We are currently assessing these newly converted murine vectors in an erythroid murine cell line, containing a large deletion of all four α-globin genes. Overall, these animal assays will determine the candidate clinical vector, and underpin the potential of this promising stem cell gene therapy as a curative approach for patients suffering from severe α-thalassemia.

Benefits to stem cell science: This research project will pave the way for the first development of a potential curative approach for α-thalassemia using the patient’s own hematopoietic stem cells. Moreover, these assays will provide further characterization of uncorrected and corrected RBC development in α-thalassemia patients, thereby shedding light on the characterization of the disease. Fundamentally, this research project will deepen our understanding and expertise on the usage of lentiviral vectors for stem cell gene addition treatments for immune system and blood disorders, such as β-thalassemia and sickle cell disease.