Transgene Expression in Dogs After Liver-Directed Hydrodynamic Delivery ofSleeping BeautyTransposons Using Balloon Catheters

Abstract

The Sleeping Beauty transposon system has been extensively tested for integration of reporter and therapeutic genes in vitro and in vivo in mice. Dogs were used as a large animal model for human therapy and minimally invasive infusion of DNA solutions. DNA solutions were delivered into the entire liver or the left side of the liver using balloon catheters for temporary occlusion of venous outflow. A peak intravascular pressure between 80 and 140 mmHg supported sufficient DNA delivery in dog liver for detection of secretable reporter proteins. Secretable reporters allowed monitoring of the time course of gene products detectable in the circulation postinfusion. Canine secreted alkaline phosphatase reporter protein levels were measured in plasma, with expression detectable for up to 6 weeks, while expression of canine erythropoietin was detectable for 7-10 days. All animals exhibited a transient increase in blood transaminases that normalized within 10 days; otherwise the treated animals were clinically normal. These results demonstrate the utility of a secreted reporter protein for real-time monitoring of gene expression in the liver in a large animal model but highlight the need for improved delivery in target tissues to support integration and long-term expression of Sleeping Beauty transposons.

Prolonged Expression of Secreted Enzymes in Dogs After Liver-Directed Delivery ofSleeping BeautyTransposons: Implications for Non-Viral Gene Therapy of Systemic Disease

“…The canine cSEAP qPCR was performed using Taqman technology, as previously described. 41 The limit of detection was 2 · 10 -4 copies per diploid genome equivalent in dog liver. Transposon and transposase-encoding plasmids.…”

“…This procedure delivered therapeutic plasmids to both right and left lobes of the liver. 41 The single-balloon system ( Fig. 1) was used in all dog experiments except Dogs 9-1 and 13.…”

“…The infusion parameters developed by Hyland et al 41 served as guidelines: DNA concentration of 2 mg/kg in a total infusion solution volume of 200 mL was delivered in 10-20 s to achieve intravascular pressure peaks of 100-200 mmHg ( Table 1). The DNA dose in all but one dog was within a 1.5fold range of 2 mg/kg, which Hyland et al found to be nearly saturating.…”

“…2. SB transposons for cSEAP expression and human (h)GUSB and hIDUA have been described previously, 13,18,19,41,45 as have plasmids for co-delivery of expression cassettes for SB transposases, pCMV-SB11, mini(m)Ub-SB11, 19,46 and pCMV-SB100X. 47 Liver-specific promoter (LSP) ApoEHCRhAAT 40 was a gift from Dr. Mark Kay (Stanford University, CA).…”

“…The injection route, properties of the vasculature and of the target organ (the liver), as well as the direction of flow after hydrodynamics-based injection are important parameters that differ substantially between mice and large animals. 4,41,42 Until recently, the efficacy of non-viral DNA delivery to treat large animals has been discouraging. 4,[42][43][44] The authors developed and examined the potential of catheter-mediated infusion via the hepatic veins wherein targeted liver tissues are isolated from the blood flow by balloon occlusion of blood vessels.…”