Graphical AbstractVerifiedLentivirus-mediated gene therapy in a beta-thalassemia mouse model
BACKGROUND: Lentiviral vector (LV)-mediated autologous hematopoietic stem cell gene therapy is expected to be a novel curative treatment for β-thalassemia. The LV serves as a core agent of gene therapy, directly influencing future clinical efficacy and treatment costs. Therefore, the primary task is to develop high-performance lentiviral vectors. OBJECTIVE: To explore the feasibility of an ex vivo gene therapy and assess the activity and functionality of the β-globin-LV in thalassemic mice. METHODS: A novel lentiviral vector, HS40-LV, carrying the human β-globin gene cassette, was constructed. 7.5 Gy-conditioned Hbbth3/+ mice were subjected to HS40-LV-modified hematopoietic stem cell transplantation. Normal mice and untreated thalassemic mice served as controls. Peripheral blood samples were collected from mice at 2, 4, 6, 8, and 10 months post-treatment. The integrated proviral DNA in the individual sample was detected by using qPCR. The proportion of red blood cells expressing human β-globin was detected by fluorescence-activated cell sorting. Fresh whole blood was collected for blood smears, which were used for Giemsa staining, reticulocyte staining, and fully automated blood cell analysis. At 10 months post-treatment, the liver, spleen, and bone marrow tissues were sampled from all three groups to prepare single-cell suspensions and extract genomic DNA for qPCR detection of vector marking; flow cytometry was used to detect cells expressing transgenic β-globin; portions of spleen and liver were subjected to hematoxylin-eosin staining and Prussian blue staining. RESULTS AND CONCLUSION: (1) The HS40-LV vector achieved a transduction efficiency of 50% in hematopoietic stem cells. (2) During the 10-month follow-up, the proportion of vector-marked cells and β-globin-positive red blood cells in peripheral blood of treated mice steadily increased, reaching an average of 50% vector marking and 70% β-globin-positive red blood cells at 10 months post-transplantation. (3) Biological distribution of the lentiviral vector and expression of transgenic β-globin were also detected in liver, spleen, and bone marrow hematopoietic tissues. (4) Gene therapy corrected hematological parameters in thalassemic mice, such as significant reductions in poikilocytes, reticulocytes, and cell fragments, and a significant increase in overall hemoglobin levels. (5) Histopathological improvements were also observed, with significant reductions in iron deposition in spleen and liver, and improved extramedullary hematopoiesis. These results indicate that the novel HS40-LV vector achieved stable expression in vivo, and modified cells corrected some symptoms in thalassemic mice.
