Analysis of Adeno-Associated Virus-Mediated ex vivo Transferred Human β-Globin Gene in Bone Marrow Engrafted Mice

Abstract

Adeno-associated virus (AAV)-2 was developed as a useful vector for human gene therapy. In this report, we analyzed the integration and expression of AAV-mediated ex vivo transferred human β-globin gene in bone marrow (BM) reconstituted mice. Recombinant AAV (rAAV) containing human β-globin gene was packaged by infecting individual G418-resistant BHK-21 cell clones integrated with the plasmid AV53HS432Δβ2.0Neo with recombinant herpes simplex virus, which can express rep and cap genes of wild-type AAV. The titer of rAAV was determined using slot blot hybridization with a result of 10¹³ virus particles/ml (genome copy number). Low-density mononuclear cells were isolated from fetal livers of embryos from pregnant C57BL/6 mice at 14–16 days of gestation and were infected with rAAV. The transduced hematopoietic cells were then reinfused into lethally irradiated C57BL/6 recipient mice via tail vein injection. To analyze the provirus in short-term and long-term BM reconstituted mice, PCR/Southern blot and RT-PCR were performed to identify the integrity of the provirus and to detect the expression of human β-globin gene, respectively. Genomic DNA was extracted from spleen nodules of BM reconstituted mice 12 days after transplantation. Human β-globin gene was detected in 1 out of 6 nodules using PCR combined with Southern blot. Human β-globin gene was also detected in the BM and thymus of mouse Y6161, in the thymus and spleen of mouse Y6162 and in the BM of mice Y6211 and Y6212. RT-PCR revealed low levels of expression of human β-globin gene in the BM of mouse Y6211. Our results suggested that the efficiency of AAV-mediated human β-globin gene integration into hematopoietic stem/progenitor cells was very low. It is necessary to perform further research on AAV biology before applying gene therapy that requires integration of a foreign gene into host chromosomes.