Lentivirus-mediated gene therapy in a beta-thalassemia mouse model

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 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.

1. Introduction

β-Thalassemia is a common inherited hematological disorder in southern China, caused by mutations in the HBB gene leading to reduced (β+) or absent (β0) β-globin synthesis. This results in accumulation of free α-globin inclusions in erythroid progenitors, causing premature cell death and ineffective erythropoiesis. Mature red blood cells undergo peripheral hemolysis, exacerbating anemia. Lifelong transfusion with iron chelation is the standard supportive care, but frequent transfusions lead to organ damage and reduced quality of life, with high medical costs (approximately 4 million RMB). Allogeneic hematopoietic stem cell transplantation is curative, but limited by donor availability, immune rejection, and graft-versus-host disease risk.

Lentiviral (LV)-mediated ex vivo gene therapy is a promising emerging strategy, offering a potential curative alternative for thalassemia patients. Autologous hematopoietic stem cells are readily available, with low risk of immune rejection and graft-versus-host disease, and ideally a single treatment could provide lifelong cure. International research teams have conducted preclinical and clinical trials with encouraging results. For instance, the French team led by Philippe Leboulch and BlueBird Bio have conducted multiple clinical trials, with most β-thalassemia patients achieving transfusion independence or reduced transfusion requirements. Other teams, such as those led by Giuliana Ferrari and Sadelain, have also reported positive clinical outcomes. In China, clinical teams led by Ouyang et al. and Li et al. have initiated small-scale trials with patients achieving transfusion independence. Currently, only BlueBird Bio's Zynteglo® has been approved for transfusion-dependent β-thalassemia, but at a high cost (approximately $2.8 million per patient). However, safety concerns have arisen: two patients with sickle cell disease treated with a similar LV developed acute myeloid leukemia, and a recent trial reported 7 cases of hematologic malignancies among 67 patients treated with another LV for cerebral adrenoleukodystrophy. These incidents underscore the need for safer and more efficient vectors.