Abstract: 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. qPCR was used to detect the labeling ratio of the LV. Flow cytometry was used to determine the proportion of cells expressing transgenic β-globin. Portions of spleen and liver tissues were fixed, embedded, sectioned, and subsequently stained with hematoxylin-eosin and Prussian blue.
RESULTS AND DISCUSSION: (1) The HS40-LV achieved a 50% transduction efficiency in hematopoietic stem cells. (2) Over the 10-month follow-up period, the proportion of vector-marked cells and β-globin-expressing cells in the peripheral blood of treated mice exhibited a sustained increase, reaching up to 50% marking efficiency and 70% β-globin-positive red blood cells. (3) The presence of the HS40-LV proviral DNA and transgenic β-globin was detected in the liver, spleen, and bone marrow. (4) Gene therapy achieved hematological correction in thalassemic mice, manifested by a significant reduction in poikilocytes, reticulocytes, and cellular debris, along with a marked increase in total hemoglobin levels. (5) Histopathological remission was also observed, including decreased iron deposition in the spleen and liver and ameliorated extramedullary hematopoiesis. These findings demonstrate that the novel HS40 LV vector achieves stable expression in vivo, and the modified cells partially relieve symptoms in thalassemia mice.

Key words: β-thalassemia, gene therapy, lentiviral vector, Hbbth3/+ thalassemia mouse, hematopoietic stem cells, erythroid-specific regulatory element, in vivo labeling, phenotype correction