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

Abstract

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 genomes͞ ml) rAAV expressing hF.IX was prepared, purified, and injected into hindlimb muscles of C57BL͞6 mice and Rag 1 mice. In the immunocompetent C57BL͞6 mice, immunof luorescence 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 ng͞ml) 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 immunof luorescent 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.

Adeno-associated virus–mediated gene transfer of endostatin inhibits angiogenesis and tumor growth in vivo

“…18,41 Skeletal muscle in particular has been used successfully for long -term expression of transgenes by rAAV. 21,30,42,43 When this approach was taken in the present investigation, it was observed that a single intramuscular injection of the rAAVHuEndo vector could lead to elevated serum endostatin levels by 4 weeks after injection (Fig 7 ). The endostatin levels reached a plateau value by 6 to 8 weeks (Fig 7) and were maintained as long as 4 months after injection.…”

Adeno-associated virus-mediated delivery of erythropoietin leads to sustained elevation of hematocrit in nonhuman primates

“…It should be noted that this decline in expression in baboons has not been seen in rodent models following intramuscular injection. 10,[12][13][14][15][16] We did not see a decline in hematocrit which corresponded to the decline in serum Epo levels. Since it has been 16 weeks (112 days) from the initial decline in Epo levels, it is unlikely that this continued elevation of hematocrit is due to the slow turnover (life span of approximately 120 days) of red blood cells.…”

“…11 There are five recent reports demonstrating the potential of sustained gene delivery following administration of rAAV vectors in mouse muscle. 10,[12][13][14][15] We have recently demonstrated the complete correction of a metabolic disease in a mouse model by a single intramuscular injection of rAAV expressing leptin. 16 In most of these reports there is evidence of long-term expression of the transgene following a single dose administration into mouse skeletal muscle.…”

Treatment of lysosomal storage disease in MPS VII mice using a recombinant adeno-associated virus

“…[18][19][20][21] In treated muscle of immunocompetent mice, this expression was shown to persist for 1.5 years. 19 Potentially therapeutic proteins that have been shown to be delivered and expressed by AAV vectors include erythropoietin, 20 cystic fibrosis transmembrane conductance regulator, 22 blood coagulation factor IX, 23,24 and GUS. 21 In none of these cases, however, were experiments designed to show that the AAV-mediated gene therapy actually affected the long-term course of a disease.…”