126. Non-Viral Gene Therapy By Liver-Directed Hydrodynamic Delivery of Sleeping Beauty Transposons to Treat Hemophilia and Mucopolysaccharidoses in Dogs

The goal of gene therapy is to achieve sustained expression of a transgene encoding an enzyme deficient in patients. Others and we have reported effectiveness of the Sleeping Beauty (SB) transposon system for gene therapy of both hemophilia B and mucopolysaccharidoses (MPS) types I and VII in adult mice. Although more than 99% of transgene expression comes from the liver following hydrodynamic delivery, restoration of deficient enzyme activity in other organs can be achieved through metabolic cross-correction whereby IDUA enters the circulation and is distributed to other tissues. However, the efficacy of hydrodynamic delivery to treat animals larger than mice has been discouraging. We have tested the use of balloon-catheters for intravascular infusion and expression of SB transposons in the liver of dogs as a large animal model for human therapy. Balloon-catheters were introduced under fluoroscopic guidance through either the jugular or the femoral vein and positioned in the inferior vena cava for occlusion of venous outflow of the and retrograde infusion through the left hepatic vein. Infusions were 10 seconds in duration and delivered volumes of DNA solution equal to about three times the estimated blood volume of the left side of the liver. Using canine secreted alkaline phosphatase (cSEAP) as a reporter we have developed effective protocols for infusion of transposons into canine liver as a scale-up for preclinical gene therapy studies in large animal models of human disease. We tested our protocol on both Factor IX (FIX)-deficient and beta-glucuronidase (GUSB)-deficient young dogs by hydrodynamically infusing two plasmids, one that harbors a T2-SB transposon containing either a canine FIX gene or a canine GUSB gene under transcriptional direction of a CAGGS or liver specific promoter (LSP) and a second carrying either the SB11 or SB100X transposase regulated by the CMV early promoter. Duration of FIX and GUSB expression in the treated dogs was transient, lasting only about 1 – 8 weeks. PCR analysis of plasmids in liver suggest that the efficiency of catheter-mediated delivery to canine livers is approximately 0.1-1% that in the mouse, which accounts for the relatively poor levels of transgene activity in the larger animals. We discuss our hypothesis that the larger vascular net with many more bifurcations in the livers of large animals lowers both impulse and shear forces necessary for effective hydrodynamic delivery in the liver.