Lentiviral vector containing beta-globin gene for beta thalassemia gene therapy

Abstract

Background and aim: Beta thalassemia is a common monogenic disorder caused by partial or complete reduction of beta globin chains synthesis. In recent years allogeneic bone marrow transplantation (BMT) has been considered to be the successful cure for patients with thalassemia major, however this is restricted due to limited number of HLA-matched donors. Therefore, molecular approaches including gene therapy for direct normal beta globin gene transmission seem quite promising to cure thalassemia. The goal of this study was to evaluate the beta globin gene expression in K562 cell line, as a model of hematopoietic cell, transduced with lentiviral vector carrying normal beta globin gene. Methods: For our purpose, we designed the DEST Lentiviral vector (3rd generation) carrying normal beta globin gene and its promoter and packaged in LentiX-293T cell line. The K562 cell line was transduced by packaged lentivirus containing β-globin cassette (pLenti-HBB). Bulk cells suspension form K562 cells were diluted and seeded into 96-well micro plate by manual pipetting. After single cell expansion, β-globin protein expression level in single cell clones was determined by flowcytometry and Western blotting. Results: Our results showed that we have successfully packaged and generated lentivirus in LentiX-293T cell line. Flowcytometry analysis showed that beta globin protein expression achieved 20.3 in bulk population of K562 cells in MOI = 20 and 74.4 in F2 clone. Conclusion: These data indicated that vector used in this study is efficient in vitro and could be applied in gene therapy in patient's hematopoietic stem cell. The final goal of this study is to examine designed vector in hematopoietic stem cells promising therapeutic strategy for beta thalassemia and sickle cell disease. © 2022 Elsevier Inc.