Microwave assisted CLARITY for whole organ surveillance

Abstract

CO RR EC TE D P RO OF transposon system for gene therapy of both MPS I and MPS 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 either IDUA or GUSB 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. Ballooncatheters were introduced under fluoroscopic guidance through either the jugular vein or the femoral vein and positioned in the inferior vena cava for occlusion of venous outflow of the retrograde infusion through the left hepatic vein. Infusions were 10 s in duration and delivered a volume of DNA solution equal to 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 the liver of dogs as a scale-up for preclinical gene therapy studies in large animal models of human disease. We tested our protocol on MPS VIIdeficient young dogs by hydrodynamically infusing two plasmids, (1) with a T2-SB transposon containing a canine GUSB gene under transcriptional direction of a CAGS or Liver Specific Promoter (LSP) and (2) with the SB100 transposase regulated by the CMV early promoter (Figure). Duration of sustained GUSB expression in the two dogs and transient increases in the levels of specific liver enzymes will be presented.