603. Correction of Hemophilia B Phenotype by Novel Method of Regional Intravenous Delivery of AAV Vector to Skeletal Muscle of Hemophilia B Dogs
Authors: Valder Arruda, Hansell Stedman, Haiyan Jian, Glenn Pierce, Joerg Schuettrumpf, Christian Furlan Freguia, Stefano Baila, Kapil Gopal, Timothy Nichols, Katherine High
Journal: Molecular Therapy (2005)

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Intramuscular (IM) injection of AAV-F.IX in humans was safe and resulted in gene transfer and expression. However, at the doses administered, it did not result in sustained circulating levels >1% of normal. Although access to the skeletal muscle can be easily obtained by direct IM injection. However, achievement of target doses in humans proved impractical because of the large number of injections required. We sought to exploit techniques that allowed widespread transduction of muscle fibers. The major obstacle to vector distribution to large areas of skeletal muscle is the vascular endothelial barrier. However, the rich network of capillaries can be chemically and/or mechanically modified to ensure vascular leakage of vectors. In hemophilia B dogs, we showed (Blood, 2005) that infusion of AAV2-canineF.IX through femoral artery in the presence of histamine to increase capillary permeability, results in long-term expression of robust therapeutic F.IX levels (4-14% normal). Here we report the application of a novel approach, afferent transvenular extravasation (ATVE), in which the vector is injected through a superficial saphenous vein in the distal part of the limb under elevated hydrostatic pressure. Briefly, a tourniquet was placed at the level of the proximal thigh. AAV2-F.IX was diluted in 20 ml of PBS/kg and infused rapidly under the pressure of 300 mmHg. The vector-solution dwells for 15 min before tourniquet release. Hemophilia B dogs treated with AAV2 at doses of 1|[times]|1012vg/kg (n=2) expressed F.IX levels of 2% (100-125ng/ml). Increasing the vector dose to 3|[times]|1012vg/kg (n=3) resulted in circulating F.IX plateau levels of 6%-23% (300-1160 ng/ml). F.IX is biologically active as judged by excellent correlation between clotting times/antigen levels and near normalization of the whole blood clotting time. A sixth dog received an AAV dose of 8.5|[times]|1012vg/kg; this dose seems to be excessive since after F.IX levels increased to 713 ng/ml (day 21) we documented a slow decrease to <100ng/ml due to low-level of a non-neutralizing antibody to F.IX. Muscle enzymes levels increased transiently in 2 of 6 dogs up to day 2 post-injection. Endothelial cell damage markers such as vWF and IL-6 assayed at several time points following vector delivery did not differ from baseline levels. Thus, ATV provides an efficient, non-invasive, and safe strategy for widespread delivery of vector to skeletal muscle in the absence of vasoactive drugs. The doses required to achieve therapeutic F.IX levels (6-23%) are similar to doses already prove to be safe in earlier clinical trials. Furthermore, ATVE abolishes the dose advantage in gene transfer of liver-directed over muscle-directed gene transfer for hemophilia B. These results establish an experimental basis for clinical studies of this delivery method in humans with hemophilia B.