[Optimization of β-globin Stable Expression Using the Third Generation Lentiviral Vector for β-thalassemia Therapy]

Abstract

Objective: To provide a research basis for a safe and effective cell therapy for β-thalassemia through optimization of HS4 region of the third generation lentiviral vector for stable expression of β-globin.

Methods: The human β-globin HS4 region in the third generation lentiviral expression vector was optimized to construct the lenti-HBB, and the transcription and translation of β-globin gene were analyzed by RT-PCR and Western blot after the transduction of lenti-HBB in MEL cell line. Furthermore, the erythroid differentiation of CD34⁺ cells which were transduced lentiviral virus carrying human β-globin from normal human umbilical cord blood cells and peripheral blood cells of patients with β-thalassemia major were confirmed by colony formation assay, cell smear assay and flow cytometry. The safety and effectiveness of the optimized lenti-HBB were verified by NSG mouse in vivo test.

Results: The human β-globin was expressed stably in the MEL cells, and CD34⁺ cells from health umbilical cord blood as well as PBMC from patient with β-thalassemia major transduced with lenti-HBB could be differentiated to mature red blood cells. The β-globin expression and differentiation in CD34⁺ cells were demonstrated successfully in the NSG mouse for about 35 months after post-transplant.

Conclusion: Stable β-globin expression through the optimization of HS4 from CD34⁺ in the third generation lentiviral vector is safe and effective for patients with severe β-thalassemia and other β-globin abnormal diseases.

Keywords: gene therapy; lentivirus; β-globin; β-thalassemia.