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904. High Efficiency Hepatic Transduction and Long-Term Transgene Expression by Delivering Helper-Dependent Adenoviral Vectors into the Surgically Isolated Liver of Nonhuman Primates
INBORN ERRORS OF METABOLISM: LIVER AND PANCREATIC DISEASES containing a potent expression cassette with a vector expressing the transgene at a lower potency. Our results suggest that vector expressing greater OTC levels allows correction of orotic acid overproduction with lower vector doses. Moreover, we were able to achieve phenotypic correction with a dose (1x1012vp/kg) which is significantly lower than those previously reported for correction with either first-generation or HDAd vectors. Vectors containing the expression cassette used in this study combined with other strategies for improving HDAd therapeutic index will likely permit application of these vectors for the treatment of OTC deficiency as well as other urea cycle disorders. There likely is a limit to the benefit of overexpressing OTC in a smaller population of deficient cells given the magnitude of nitrogen transfer that must be handled by the liver on a daily basis. At the same time, we will need to evaluate potential adverse consequences of over-expression of these enzymes.
904. High Efficiency Hepatic Transduction and Long-Term Transgene Expression by Delivering Helper-Dependent Adenoviral Vectors into the Surgically Isolated Liver of Nonhuman Primates Nicola Brunetti-Pierri,1 Thomas Ng,2 David Iannitti,2 William Cioffi,2 Donna Palmer,1 Arthur Beaudet,1 Milton Finegold,3 Dee Carey,4 Philip Ng.1 1 Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, TX; 2Department of Surgery, Brown Medical School, Providence, RI; 3Department of Pathology, Baylor College of Medicine, Houston, TX; 4Southwest Foundation for Biomedical Research, San Antonio, TX. Helper-dependent adenoviral vectors (HDAd) are attractive vectors for liver-directed gene therapy because they can mediate long-term, high level transgene expression with no long-term toxicity. However, as a consequence of a threshold effect, high vector doses are required to achieve efficient hepatic transduction by peripheral intravenous injection which unfortunately results in a dosedependent activation of the innate inflammatory response. Clearly strategies to overcome the threshold to efficient hepatic transduction are needed to improve the therapeutic index of HDAd. We hypothesized that this obstacle could be surmounted by delivering the vector exclusively to the liver. To test this hypothesis, we have injected HDAd directly into the surgically isolated liver via the portal vein in nonhuman primates. Total hepatic isolation was achieved by occluding hepatic inflow from the portal vein and hepatic artery and by occluding hepatic venous outflow at the inferior vena cava. Prior to total hepatic isolation, saline was infused into the portal vein to flush blood out of the liver. The vector was then injected directly into the liver via the portal vein and allowed to dwell for 30 minutes, following which unabsorbed vector was flushed out via a catheter placed in the vena cava to minimize systemic vector dissemination. Our results revealed that significantly higher hepatic transduction efficiencies can be achieved with relatively low vector doses compared to peripheral intravenous injection. Importantly, stable, high levels of transgene expression were observed for up to one year with no long-term toxicity. This approach may increase the safety and efficacy of HDAd-mediated, liver-directed gene therapy by minimizing the dose required to achieve efficient hepatic transduction and by minimizing systemic vector dissemination.
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905. Hydrodynamic Injection of HelperDependent Adenoviral Vectors Increases Liver Transduction Efficiency and Decreases Acute Inflammatory Response Nicola Brunetti-Pierri,1 Viraj Mane,1 Donna Palmer,1 Arthur Beaudet,1 Milton Finegold,2 Philip Ng.1 1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2Department of Pathology, Baylor College of Medicine, Houston, TX. Hydrodynamic injection of helper-dependent adenoviral vectors (HDAd) in mice results in increased hepatic transduction resulting in higher levels of long-term transgene expression compared to conventional injection. Furthermore, hydrodynamic injection resulted in reduced splenic and pulmonary transduction and reduced serum levels of the proinflammatory cytokines IL-6 and IL-12. Interestingly, hydrodynamic injection does not appear to alter Kupffer cell uptake of the vector compared to conventional injection. Taken together, these results strongly suggest that hepatic transduction by HDAd, at least alone, does not necessarily provoke a severe innate inflammatory response, but that systemic vector dissemination may play a major role in the severity of the innate inflammatory response. These results further suggest that the safety and efficacy of HDAd-mediated, liver-directed gene therapy may be improved if the vector could be preferentially, if not exclusively, targeted to hepatocytes.
906. High Efficiency Hepatic Transduction with Minimal Toxicity Following “Hydrodynamic” Injection of HDAd into Nonhuman Primates Nicola Brunetti-Pierri,1 Charles Mullins,2 Donna Palmer,1 Milton Finegold,3 Arthur Beaudet,1 Karen Rice,4 Dee Carey,4 Philip Ng.1 1 Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, TX; 2Department of Pediatics Cardiology, Baylor College of Medicine, Houston, TX; 3Department of Pathology, Baylor College of Medicine, Houston, TX; 4Southwest Foundation for Biomedical Research, San Antonio, TX. Helper-dependent adenoviral vectors (HDAd) are attractive vectors for liver-directed gene therapy because they can mediate sustained, high level transgene expression with no long-term toxicity. However, as a consequence of a threshold effect, high vector doses are required to achieve efficient hepatic transduction by peripheral intravenous injection which unfortunately results in a dosedependent activation of the innate inflammatory response. Clearly strategies to overcome the threshold to efficient hepatic transduction are needed to improve the therapeutic index of HDAd. We have recently shown that systemic hydrodynamic injection of HDAd into mice resulted in exceedingly high levels of hepatic transduction and reduction in both systemic vector dissemination and acute proinflammatory cytokines compared to conventional injection. Hydrodynamic injection offers a method of dramatically increasing the therapeutic index of HDAd but, unfortunately, cannot be applied to larger animals due to the large injection volume. Therefore, we have developed a minimally invasive method to mimic hydrodynamic injection in nonhuman primates which does not require injection of large volumes. This method involves the use of balloon occlusion catheters percutaneously positioned in the vena cava to transiently obstruct hepatic venous outflow to increase the intrahepatic pressure prior to simple peripheral intravenous injection of HDAd in a small volume. We demonstrate in nonhuman primates that this novel method of vector delivery results in an exceedingly high and unprecedented level of hepatic transduction with low vector doses resulting in stable, long-term transgene expression. This was accompanied by reduced systemic vector dissemination and minimal, transient elevations of proinflammatory cytokines. Despite the Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
904. High Efficiency Hepatic Transduction and Long-Term Transgene Expression by Delivering Helper-Dependent Adenoviral Vectors into the Surgically Isolated Liver of Nonhuman Primates Nicola Brunetti-Pierri,1 Thomas Ng,2 David Iannitti,2 William Cioffi,2 Donna Palmer,1 Arthur Beaudet,1 Milton Finegold,3 Dee Carey,4 Philip Ng.1 1 Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, TX; 2Department of Surgery, Brown Medical School, Providence, RI; 3Department of Pathology, Baylor College of Medicine, Houston, TX; 4Southwest Foundation for Biomedical Research, San Antonio, TX. Helper-dependent adenoviral vectors (HDAd) are attractive vectors for liver-directed gene therapy because they can mediate long-term, high level transgene expression with no long-term toxicity. However, as a consequence of a threshold effect, high vector doses are required to achieve efficient hepatic transduction by peripheral intravenous injection which unfortunately results in a dosedependent activation of the innate inflammatory response. Clearly strategies to overcome the threshold to efficient hepatic transduction are needed to improve the therapeutic index of HDAd. We hypothesized that this obstacle could be surmounted by delivering the vector exclusively to the liver. To test this hypothesis, we have injected HDAd directly into the surgically isolated liver via the portal vein in nonhuman primates. Total hepatic isolation was achieved by occluding hepatic inflow from the portal vein and hepatic artery and by occluding hepatic venous outflow at the inferior vena cava. Prior to total hepatic isolation, saline was infused into the portal vein to flush blood out of the liver. The vector was then injected directly into the liver via the portal vein and allowed to dwell for 30 minutes, following which unabsorbed vector was flushed out via a catheter placed in the vena cava to minimize systemic vector dissemination. Our results revealed that significantly higher hepatic transduction efficiencies can be achieved with relatively low vector doses compared to peripheral intravenous injection. Importantly, stable, high levels of transgene expression were observed for up to one year with no long-term toxicity. This approach may increase the safety and efficacy of HDAd-mediated, liver-directed gene therapy by minimizing the dose required to achieve efficient hepatic transduction and by minimizing systemic vector dissemination.
S350
905. Hydrodynamic Injection of HelperDependent Adenoviral Vectors Increases Liver Transduction Efficiency and Decreases Acute Inflammatory Response Nicola Brunetti-Pierri,1 Viraj Mane,1 Donna Palmer,1 Arthur Beaudet,1 Milton Finegold,2 Philip Ng.1 1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2Department of Pathology, Baylor College of Medicine, Houston, TX. Hydrodynamic injection of helper-dependent adenoviral vectors (HDAd) in mice results in increased hepatic transduction resulting in higher levels of long-term transgene expression compared to conventional injection. Furthermore, hydrodynamic injection resulted in reduced splenic and pulmonary transduction and reduced serum levels of the proinflammatory cytokines IL-6 and IL-12. Interestingly, hydrodynamic injection does not appear to alter Kupffer cell uptake of the vector compared to conventional injection. Taken together, these results strongly suggest that hepatic transduction by HDAd, at least alone, does not necessarily provoke a severe innate inflammatory response, but that systemic vector dissemination may play a major role in the severity of the innate inflammatory response. These results further suggest that the safety and efficacy of HDAd-mediated, liver-directed gene therapy may be improved if the vector could be preferentially, if not exclusively, targeted to hepatocytes.
906. High Efficiency Hepatic Transduction with Minimal Toxicity Following “Hydrodynamic” Injection of HDAd into Nonhuman Primates Nicola Brunetti-Pierri,1 Charles Mullins,2 Donna Palmer,1 Milton Finegold,3 Arthur Beaudet,1 Karen Rice,4 Dee Carey,4 Philip Ng.1 1 Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, TX; 2Department of Pediatics Cardiology, Baylor College of Medicine, Houston, TX; 3Department of Pathology, Baylor College of Medicine, Houston, TX; 4Southwest Foundation for Biomedical Research, San Antonio, TX. Helper-dependent adenoviral vectors (HDAd) are attractive vectors for liver-directed gene therapy because they can mediate sustained, high level transgene expression with no long-term toxicity. However, as a consequence of a threshold effect, high vector doses are required to achieve efficient hepatic transduction by peripheral intravenous injection which unfortunately results in a dosedependent activation of the innate inflammatory response. Clearly strategies to overcome the threshold to efficient hepatic transduction are needed to improve the therapeutic index of HDAd. We have recently shown that systemic hydrodynamic injection of HDAd into mice resulted in exceedingly high levels of hepatic transduction and reduction in both systemic vector dissemination and acute proinflammatory cytokines compared to conventional injection. Hydrodynamic injection offers a method of dramatically increasing the therapeutic index of HDAd but, unfortunately, cannot be applied to larger animals due to the large injection volume. Therefore, we have developed a minimally invasive method to mimic hydrodynamic injection in nonhuman primates which does not require injection of large volumes. This method involves the use of balloon occlusion catheters percutaneously positioned in the vena cava to transiently obstruct hepatic venous outflow to increase the intrahepatic pressure prior to simple peripheral intravenous injection of HDAd in a small volume. We demonstrate in nonhuman primates that this novel method of vector delivery results in an exceedingly high and unprecedented level of hepatic transduction with low vector doses resulting in stable, long-term transgene expression. This was accompanied by reduced systemic vector dissemination and minimal, transient elevations of proinflammatory cytokines. Despite the Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy