Abstract:

Objective To investigate the transfection of recombinant adeno-associated virus 2 (rAAV2) in human hematopoietic stem cells of patients with βthalassemia, and explore the feasibility of ex vivo gene therapy for β-thalassemia. Methods Six BALB/c nude mice pretreated with X-ray were divided into rAAV2-transfected group (n=4) and mock-transfected group (n=2). Isolated human hematopoietic cells from fetus with β-thalassemia major of β41-42/β654 heterozygote were infected or mock infected with rAAV2-β-globin (MOI＝50), and were transplanted into BALB/c nude mice. After transfection for 28 d and 70 d, mice were sacrificed, RT-PCR and allele-specific PCR (ASPCR) were applied to detect the expression of rAAV2-mediated β-globin gene in mouse bone marrow cells, and the levels of β-globin chains in peripheral blood of recipient mice were quantified by HPLC. Results ①The expression of human β-actin and β-globin gene was detected in all recipient mice by RT-PCR. ②By ASPCR, the expression of β41-42 was found in all recipient mice, while β654 in recipient mice on day 70. There was expression of normal β-globin gene mainly from β654 gene in both groups of mice on day 28. However, on day 70,  rAAV-2 mediated normal β-globin gene expression was found only in transfected mice.  ③The human β/α in peripheral blood of transfected mice were 0.328 and 0.325 on 70 d, 144.78% and 142.54% higher than that of mock-transfected mice (0.135). Conclusion rAAV2 vectors genetically modified hematopoietic cells of patients with β-thalassemia major could mediate long-term stable expression of normal β-globin gene in vivo, with significantly elevated expression of β-chains in human erythroid cells in peripheral blood.

Key words: recombinant adeno-associated virus, β-thalassemia, human hematopoietic cells, gene therapy