Abstract
We sought to determine whether intramuscular injection of a recombinant adeno-associated virus (rAAV) rector expressing human factor IX (hF.IX) could direct expression of therapeutic levels of the transgene in experimental animals. High titer (1012-1012 vector genomes/ml) rAAV expressing hF.IX was prepared, purified, and injected into hindlimb muscles of C57BL/6 mice and Rag 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 interstitial 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 can be used to direct stable expression of therapeutic levels of F.IX after intramuscular injection and is a feasible strategy for treatment of patients with hemophilia B.
Original language | English |
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Pages (from-to) | 5804-5809 |
Number of pages | 6 |
Journal | Proceedings of the National Academy of Sciences of the United States of America |
Volume | 94 |
Issue number | 11 |
DOIs | |
State | Published - 27 May 1997 |