Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus

作者：Roland W. Herzog, J. Nathan Hagstrom, Szu-Hoo Kung, Shing Jen Tai, James M. Wilson, Krishna J. Fisher, Katherine A. High · 发表于：Proceedings of the National Academy of Sciences · 年份：1997 · DOI：10.1073/pnas.94.11.5804 · 被引用次数：477 · 研究领域：Virus-based gene therapy research、CAR-T cell therapy research、Viral Infectious Diseases and Gene Expression in Insects

We sought to determine whether intramuscular injection of a recombinant adeno-associated virus (rAAV) vector expressing human factor IX (hF.IX) could direct expression of therapeutic levels of the transgene in experimental animals. High titer (10(12)-10(13) vector genomes/ml) rAAV expressing hF.IX was prepared, purified, and injected into hindlimb muscles of C57BL/6 mice and Rag 1 mice. In the immunocompetent C57BL/6 mice, immunofluorescence staining of muscle harvested 3 months after injection demonstrated the presence of hF.IX protein, and PCR analysis of muscle DNA was positive for AAV DNA, but no hF.IX was detected in mouse plasma. Further studies showed that these mice had developed circulating antibodies to hF.IX. In follow-up experiments in Rag 1 mice, which carry a mutation in the recombinase activating gene-1 and thus lack functional B and T cells, similar results were seen on DNA analysis of muscle, but these mice also demonstrated therapeutic levels (200-350 ng/ml) of F. IX in the plasma. The time course of F.IX expression demonstrates that levels gradually increase over a period of several weeks before reaching a plateau that is stable 6 months after injection. In other experiments we demonstrate colocalization of hF.IX and collagen IV in intersitial spaces between muscle fibers. Collagen IV has recently been identified as a F.IX-binding protein; this finding explains the unusual pattern of immunofluorescent staining for F.IX shown in these experiments. Thus rAAV ...