Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5 × 10 12 vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.

Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (FIX). Using adeno-associated virus (AAV)-mediated, liver-directed gene therapy, we achieved long-term (> 17 months) substantial correction of canine hemophilia B in 3 of 4 animals, including 2 dogs with an FIX null mutation. This was accomplished with a comparatively low dose of 1 ؋ 10 12 vector genomes/kg. Canine FIX (cFIX) levels rose to 5% to 12% of normal, high enough to result in nearly complete phenotypic correction of the disease. Activated clotting times and whole blood clotting times were normalized, activated partial thromboplastin times were substantially reduced, and anti-cFIX was not detected. The fourth animal, also a null mutation dog, showed transient expression (4 weeks), but subsequently developed neutralizing anti-cFIX (inhibitor IntroductionHemophilia B is a sex-linked bleeding disorder caused by a deficiency of functional coagulation factor IX (FIX). Current replacement therapy consists of intravenous infusion of protein concentrate. However, this treatment is costly and inconvenient and carries with it the risk of blood-borne disease transmission. Furthermore, bleeds are often treated only after they have occurred, rather than prophylactically, so that chronic joint damage occurs and the risk of a fatal bleed is always present. Hemophilia is an ideal model for gene therapy because precise regulation and tissue-specific transgene expression are not required. 1,2 A number of animal models are available including knockout mice and well-described hemophilic dog colonies with phenotypes corresponding to the human disease. [3][4][5] Clinical end points for treatment are well defined. An increase of factor levels to more than 1% will improve the phenotype of the disease from severe to moderate, with reduced frequency of spontaneous bleeds, and a further increase to more than 5% will result in a mild phenotype; that is, patients would likely require factor infusion only after severe injury or during surgery. Currently the most serious complication of treatment is the formation of inhibitory antibodies to the deficient protein, which occurs with a frequency of 3% to 4% in patients with hemophilia B. 6,7 Inhibitor formation is observed mostly in those patients with extensive loss of FIX coding information. 6,8 Sustained expression of canine FIX (cFIX) in dogs with a missense mutation has been observed following administration of an adeno-associated virus (AAV) vector into the portal vein for hepatic gene transfer or into skeletal muscle. 9-11 The latter approach is currently being tested in a phase 1 clinical trial. 12 AAV vectors can be produced in a helper virus-free system, are devoid of any viral gene products, and often fail to activate antigen-specific cytotoxic T lymphocytes. 13 However, inhibitor formation is still a frequent complication following intramuscular administration of AAV vector in hemophilia B mice (with a large F9 gene deletion) and dogs with a FIX null mutation. 14,15 In these anima...