| Objective:β-thalassemia, one of the most common monogenetic disease in the world, is caused by mutation or deletion inβ-globin gene cluster that lead to decrease or absence of theβ-globin chain, as well as chronic hemolytic anemia. Till now, there is still no satisfactory treatment available forβ-thalassemia. Theoretically,β-thalassemia is curable by vector-mediated gene transfer into hematopoietic stem cells of patients withβ-thalassemia. Thus the upregulation of normalβ-globin could reestablish the balance betweenαand βchains in human erythroid cells. We have reported successful AAV-mediated globin gene transfer and long-term gene expression in murine or human hematopoietic cells. In this study, we try to genetically modify hematopoietic cells from the aborted β-thalassemia major fetus with recombinant AAV2 and explore the expression ofβ-globin gene in vivo following AAV-mediated gene transfer, which provides further support for the feasibility of AAV vectors in gene therapy forβ-thalassemia.Contents:1. The method of packaging and purifying of rAAV2-β-globin In our study, the plasmid pMT-2 including miniLCR andβ-globin gene was first identified by restriction enzyme digestion. And the calcium phosphate precipitate formed by mixing pAAV-RC, pHelper and pMT-2 was added to HEK293 cells; The packaged recombinant rAAV2-β-globin was purified with a single-step gravity-flow column; The titers of the purified stocks ranged within 1010vg/ml as determined by quantitative DNA dot blots; And sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to evaluate the high purity of recombinant virus.2. The expression of rAAV2-mediatedβ-globin gene in MEL cells in vitroMEL cells were infected or mock-infected by rAAV2-β-globin vectors in vitro. The results of PCR and RT-PCR showed that transferred humanβ-globin gene was transcriptionally active in infected MEL cells, while negative in mock-infected MEL cells, indicating that AAV could efficiently transfect erythroid cells and mediate the expression ofβ-globin transgene in vitro.3. rAAV2-mediatedβ-globin gene transfer into hematopoietic cells from abortedβ-thalassemia major fetus of homozygote typeOn the basis of the study in vitro, hematopoietic cells isolated from the abortedβ-thalassemia major fetus ofβ41-42 homozygote were transfected with rAAV2-β-globin (MOI=10) and transplanted in to 4 BALB/c-nu/nu mice pretreated with cyclophosphamide through tail intravenous injection (IV). Expression of AAV-mediated normal (3-globin gene was confirmed by allele-specific PCR in all recipient mice detected on 14d or 27d post-transplanted, as well as the mutated expression of (341/42 globin gene. The results demonstrate that recombinant AAV2 vectors could efficiently introduce and express the (3-globin transgene in human hematopoietic cells fromβ-thalassemia fetus of homozygote in vivo.4. rAAV2-mediatedβ-globin gene transfer into hematopoietic cells from abortedβ-thalassemia major fetus of heterozygote typeTo investigate the transduction efficiency of AAV vectors in patients with prevalent heterozygousβ-thalassemia, hematopoietic cells were isolated from five abortedβ-thalassemia major fetus with the genotype ofβ41-42/β71-72,β654/deletion,β41-42/β654,β41-42/pE andβ41-42/β645. Hematopoietic cells were transfected with rAAV2 (MOI=10 or 50), following transplanted into BALB/c-nu/nu mice pretreated with X-ray. The recipient mice were sacrificed on different time points to examine theβ-globin transgene expression. After transplantation, human (3-actin gene expression could be detected in both bone marrow and peripheral blood, indicating successful engraftment of human hematopoietic cells in recipient mice; Though the study in p654/deletion sample (MOI=10) failed to demonstrate AAV-mediated gene transfer due to the normal expression of mutated p654 gene and the low transgene expression, the results from other four samples identified the expression of humanβ-globin gene mediated by AAV vectors (up to 70 days). And we used rigorous HPLC method to quantify the (3-chains in human erythroid cells in vivo. The data showed that increased synthesis ofβchains could be achieved with the transduction of AAV vectors both at 10 and 50 MOI. Moreover, the higher MOI of AAV contributes to enhanced transfection efficiency and stability of transgene expression.ConclusionRecombinant AAV2 vectors can efficiently transduce hematopoietic cells from aborted (3-thalassemia major fetus. The AAV2-transduced hematopoietic cells could mediate long-term and stable expression of normal (3-globin gene in BALB/c-nu/nu mice, and significantly elevate the expression ofβ-chains in human erythroid cells in peripheral blood.
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