A gene-deleted adenoviral vector results in phenotypic correction of canine hemophilia B without liver toxicity or thrombocytopenia

Abstract

Many approaches for treating hemophilia via gene transfer have been attempted in large animal models but all have potential drawbacks. Recombinant adenoviral vectors offer high-efficiency transfer of an episomal vector but have been plagued by the cytotoxicity/immunogenicity of early-generation vectors that contain viral genes. In our current study, we have used a nonintegrating helper-dependent (HD) adenoviral vector for liver-directed gene transfer to achieve hemostatic correction in a dog with hemophilia B. We measured plasma canine factor IX (cFIX) concentrations at a therapeutic range for up to 2.5 months and normalization of the whole blood clotting time (WBCT) for about a month. This was followed by a decrease and stabilized partial correction for 4.5 months. Hepatic gene transfer of a slightly lower dose of the HD vector resulted in WBCTs that were close to normal for 2 weeks, suggesting a dose threshold effect in dogs. In sharp contrast to other studies using first-or second-generation adenoviral vectors, we ob-

Large-Scale Production of High-Quality Helper-Dependent Adenoviral Vectors Using Adherent Cells in Cell Factories

“…Additionally, a number of modified Cre-based (Barjot et al, 2002;Sakhuja et al, 2003) and FLP-based (Ng et al, 2001) systems have been developed for HDAd production. All of these systems, however, are constrained by their technical complexities (especially for production of larger quantities) and are limited to the production of modest amounts of HDAds suitable for small animal experiments or low-dose large animal studies (Morral et al, 1999;Ehrhardt et al, 2003;Brown et al, 2004). Although results from studies using this approach have been encouraging, improved methods to permit efficient and reliable production of large quantities of HDAds are needed to fully evaluate the safety and efficacy of these vectors in preclinical studies with large animal models and, ultimately, for human clinical evaluation.…”

Gutless adenovirus: last-generation adenovirus for gene therapy

“…68 Gutless vectors have been shown to express non-immunogenic transgenes during the whole life of the mouse, while expression driven by first-generation adenovirus lasted at most for 3 months. [80][81][82] Therapeutic genes carried by gutless vectors have been administered to the liver of mouse models for different diseases, such as hemophilia A and B, 83,84 obesity, 66 familial hypercholesterolemia, 81,85-87 ornithine transcarbamylase deficiency, 88 diabetes 89 and chronic viral hepatitis. 90,91 Therapeutic levels of most proteins have been documented for a long-term duration.…”

“…90,92 The encouraging results obtained in rodents had promoted the preclinical studies in larger animal models. This is the case for hemophilia A and B dogs, 72,83 which resulted in minimal acute liver toxicity and transient expression of the transgene allowing for partial or complete correction of hemophilia. As in rodents, the use of liver-specific promoters avoided the development of neutralizing antibodies against the therapeutic protein, although the expression was lower than with a viral ubiquitous promoter.…”

Liver gene therapy: advances and hurdles

“…After liver directed gene transfer in various large animal models, sustained expression of a therapeutic protein was achieved. 30,31 However, the difficulties to prepare large batches of such vectors with a clinical grade may still limit the clinical application of this approach.…”