Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector

Abstract

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.

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

“…Indeed the experience with AAV vectors injected into skeletal muscle of small or large immunocompetent animals has shown that transgene expression may persist for years. 21,44 Rather, two aspects of AAV biology are believed to contribute to the prolongation of transgene expression. The vector genome replicates as a large concatemer, either existing stably as a large episome or integrating randomly into the host genome.…”

Convection-Enhanced Delivery of AAV Vector in Parkinsonian Monkeys; In Vivo Detection of Gene Expression and Restoration of Dopaminergic Function Using Pro-drug Approach

“…Forty T225 flasks were seeded with 2.5 ϫ 10 6 cells each and grown for 3 days prior to transfection to 70 -80% confluency (approximately 1.5 ϫ 10 7 cells per flask). The transfection and purification methods (16,30) were employed for AAV production, with minor modifications. The vector production process involved cotransfection of HEK 293 cells with 20 g of each of the following three plasmids per flask: the AAV-AADC plasmid, the AAV helper plasmid (pHLP19, containing the AAV rep and cap genes), and the adenovirus helper plasmid (pladeno-5, previously known as pVAE2AE4-2 and composed of the E2A, E4, and VA RNA genes derived from purified adenovirus-2), using the calcium phosphate method for a period of 6 h. After transfection, the media were replaced and the cells were harvested 3 days later.…”

Major role of local immune responses in antibody formation to factor IX in AAV gene transfer

“…2 Our published data demonstrate sustained long-term F.IX expression via adeno-associated viral (AAV) vector-mediated gene transfer to the liver or skeletal muscle, and the safety of both approaches has been tested in Phase I clinical trials. [2][3][4][5] AAV serotype 2 vectors are based on a nonpathogenic, replication defective parvovirus with a 4.7-kb single-stranded DNA genome. Advantages of this vector include absence of viral coding sequences, low innate immunogenicity, production without helper virus, and efficient in vivo gene transfer to nondividing target cells (eg hepatocytes or muscle fibers) with mostly episomal persistence of the vector genome.…”

“…administration, which did not require immune suppression unless very high vector doses were injected. 4,11 After comparing vector doses per site of injection between different treated hemophilia B dogs and experiments with a more efficient AAV serotype-1 vector, we suspected local F.IX antigen doses in transduced muscle to be a critical parameter for the risk of inhibitor formation. 7,11 Based on these observations, only subjects with F.IX missense mutations were enrolled in the trial based on muscle delivery, and the vector dose per site of i.m.…”