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986. Balloon Occlusion Catheter-Based Delivery of HDAd Permits Efficient Hepatic Transduction Using Clinically Relevant Low Vector Doses in Nonhuman Primates
Inborn Errors of Metabolism had heights of lumbar and cervical vertebrae that were only 61% of normal and femurs that were only 87% of normal. RV-treated dogs had vertebral heights that were 83% of normal and femurs that were 99% of normal, (p<0.02 for all comparisons), demonstrating a significant improvement in bone lengths. Radiographs of RV-treated dogs at 7 years show some degenerative joint disease, but were much improved when compared with those in 2 year-old untreated MPS VII dogs. The corneas of the RV-treated dogs remain remarkably clear. None of the dogs have evidence of neoplasia. Neonatal IV injection of RV continues to be an extremely promising approach for treating MPS VII. We are currently making a self-inactivating RV vector to reduce the chance of insertional mutagenesis.
985. Biodistribution of AAV1-LPLS447X Vector Co-Administered with Immunosuppression to Lipoprotein Lipase Deficient Patients in a Phase II Study Daniel Gaudet,1 Julie Méthot,1 Christiane Essiembre,1 Diane Brisson,1 Sander van Deventer,2 Annemarie Kleefstra,2 Janneke Meulenberg.2 1 Community Genomic Medicine Center and Lipid Clinic, Chicoutimi, QC, Canada; 2AMT, Amsterdam, Netherlands.
Lipoprotein Lipase (LPL) deficiency is a disease characterized by the presence of marked chylomicronemia. Chylomicrons are triglyceride-rich lipoproteins and their accumulation is caused by deficiency of LPL, the principal enzyme involved in the clearance of triglycerides from plasma. Humans who are deficient in LPL usually present in childhood with repetitive bouts of colicky pain and typically, failure to thrive, growth retardation, and eruptive xanthomas. The most severe complication, however, is acute hemorrhagic pancreatitis, which can be lethal. There is no adequate treatment for LPL deficiency. We have developed a gene therapy strategy to restore LPL function and consequently enhance triglyceride metabolism. This gene therapy strategy is based on AAV1-LPLS447X, an adenoassociated viral vector (AAV) pseudotyped with serotype 1 capsids expressing human LPLS447X. AAV1-LPLS447X is delivered to skeletal muscle, a site of endogenous LPL expression. The AAV1-LPLS447X vector was initially produced by co-transfection of a helper and vector plasmid in human embryonic kidney cells (HEK293) and designated AMT-010. The safety and efficacy of this vector was successfully tested in LPL deficient patients in a Phase I/II study. A major drawback of the plasmid transfection method, however, is that it is laborintensive, costly and not scalable. Therefore, AMT has developed a new production system making use of baculoviruses. Infection of insect cells in suspension culture with recombinant baculoviruses containing the essential genes for AAV vector production eliminates the transfection process, and thereby resulted in a highly scalable and efficient production method. AMT-011, the AAV1-LPLS447X vector produced with this optimized production system was tested in a subsequent Phase II study in LPL deficient subjects. Two patients received 3x1011 genome copies (gc)/kg AMT-011, four patients received 3x1011 gc/kg AMT-011 and immunosuppression, and eight patients received 1x1012 gc/kg AMT-011 and immunosuppression. Immunosuppression was co-administered to inhibit cellular immune responses to AAV1 capsid. Serum, saliva, urine, semen, and muscle biopsies were collected to analyze the presence of vector sequences using a sensitive quantitative PCR. The levels of AMT-011 vector DNA in these tissues will be presented and compared with those collected for AMT-010 in a previous phase I/II study.
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
986. Balloon Occlusion Catheter-Based Delivery of HDAd Permits Efficient Hepatic Transduction Using Clinically Relevant Low Vector Doses in Nonhuman Primates
Nicola Brunetti-Pierri,1 Gary Stapleton,2 Mark Law,2 Donna Palmer,1 Arthur Beaudet,1 Charles Mullins,2 Philip Ng.1 1 Human and Molecular Genetics, Baylor College of Medicine, Houston, TX; 2Pediatric Cardiology, Baylor College of Medicine, Houston, TX.
Helper-dependent adenoviral vectors (HDAds) hold tremendous potential for liver-directed gene therapy by providing long-term transgene expression without chronic toxicity. The main obstacle hindering clinical application of these vectors is acute toxicity which is dose-dependent. In the case of humans, a systemic dose of 6x1011 vp/ kg of an earlier generation Ad vector was lethal in one of two partial OTC-deficient patients whereas a ½-log lower dose (2x1011 vp/kg) was well tolerated. Therefore, in considering clinical Ad-mediated liver-directed gene therapy, administration of lower doses is critical for safety. Unfortunately, while safe, systemic intravenous (IV) injection of lower doses is not efficacious because they yield little to no hepatocyte transduction. Therefore, it is important to develop novel strategies to increase the efficiency of hepatocyte transduction with low vector doses. Towards this goal, we have developed in nonhuman primates a minimally invasive method to achieve efficient hepatic transduction using low and safe HDAd doses. Briefly, a balloon catheter was percutaneously positioned in the inferior vena cava to occlude hepatic venous outflow and an HDAd expressing the baboon a-fetoprotein (bAFP) reporter was injected into the liver via a hepatic artery catheter and this method was compared to simple systemic peripheral IV injection. In these experiments, two baboons were injected with 3x1010 vp/kg HDAd by simple systemic peripheral IV injections (Fig. solid and open squares). As expected, at this low dose little to no transgene expression was detected. In contrast, when the same dose of 3x1010 vp/kg was administered to two baboons using the balloon occlusion catheter based approach (Fig. solid and open circles), an increased level of transgene expression of up to 80-fold was observed compared to systemic peripheral IV injection. In addition, transgene expression persisted at this high level for the duration of the observation period of at least 56 days. More importantly, when an even lower dose of 1x1010 vp/kg was administered to two baboons by the balloon occlusion catheter approach (Fig. solid and open triangles), the level of transgene expression was 30-fold higher than systemic peripheral IV injection of the higher 3x1010 vp/kg dose (Fig. solid and open squares). Thus, the balloon occlusion catheter technique permits delivery of clinically relevant low HDAd doses to achieve efficient hepatic transduction and may be a first step towards clinical application of HDAd for liver-directed gene therapy.
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985. Biodistribution of AAV1-LPLS447X Vector Co-Administered with Immunosuppression to Lipoprotein Lipase Deficient Patients in a Phase II Study Daniel Gaudet,1 Julie Méthot,1 Christiane Essiembre,1 Diane Brisson,1 Sander van Deventer,2 Annemarie Kleefstra,2 Janneke Meulenberg.2 1 Community Genomic Medicine Center and Lipid Clinic, Chicoutimi, QC, Canada; 2AMT, Amsterdam, Netherlands.
Lipoprotein Lipase (LPL) deficiency is a disease characterized by the presence of marked chylomicronemia. Chylomicrons are triglyceride-rich lipoproteins and their accumulation is caused by deficiency of LPL, the principal enzyme involved in the clearance of triglycerides from plasma. Humans who are deficient in LPL usually present in childhood with repetitive bouts of colicky pain and typically, failure to thrive, growth retardation, and eruptive xanthomas. The most severe complication, however, is acute hemorrhagic pancreatitis, which can be lethal. There is no adequate treatment for LPL deficiency. We have developed a gene therapy strategy to restore LPL function and consequently enhance triglyceride metabolism. This gene therapy strategy is based on AAV1-LPLS447X, an adenoassociated viral vector (AAV) pseudotyped with serotype 1 capsids expressing human LPLS447X. AAV1-LPLS447X is delivered to skeletal muscle, a site of endogenous LPL expression. The AAV1-LPLS447X vector was initially produced by co-transfection of a helper and vector plasmid in human embryonic kidney cells (HEK293) and designated AMT-010. The safety and efficacy of this vector was successfully tested in LPL deficient patients in a Phase I/II study. A major drawback of the plasmid transfection method, however, is that it is laborintensive, costly and not scalable. Therefore, AMT has developed a new production system making use of baculoviruses. Infection of insect cells in suspension culture with recombinant baculoviruses containing the essential genes for AAV vector production eliminates the transfection process, and thereby resulted in a highly scalable and efficient production method. AMT-011, the AAV1-LPLS447X vector produced with this optimized production system was tested in a subsequent Phase II study in LPL deficient subjects. Two patients received 3x1011 genome copies (gc)/kg AMT-011, four patients received 3x1011 gc/kg AMT-011 and immunosuppression, and eight patients received 1x1012 gc/kg AMT-011 and immunosuppression. Immunosuppression was co-administered to inhibit cellular immune responses to AAV1 capsid. Serum, saliva, urine, semen, and muscle biopsies were collected to analyze the presence of vector sequences using a sensitive quantitative PCR. The levels of AMT-011 vector DNA in these tissues will be presented and compared with those collected for AMT-010 in a previous phase I/II study.
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
986. Balloon Occlusion Catheter-Based Delivery of HDAd Permits Efficient Hepatic Transduction Using Clinically Relevant Low Vector Doses in Nonhuman Primates
Nicola Brunetti-Pierri,1 Gary Stapleton,2 Mark Law,2 Donna Palmer,1 Arthur Beaudet,1 Charles Mullins,2 Philip Ng.1 1 Human and Molecular Genetics, Baylor College of Medicine, Houston, TX; 2Pediatric Cardiology, Baylor College of Medicine, Houston, TX.
Helper-dependent adenoviral vectors (HDAds) hold tremendous potential for liver-directed gene therapy by providing long-term transgene expression without chronic toxicity. The main obstacle hindering clinical application of these vectors is acute toxicity which is dose-dependent. In the case of humans, a systemic dose of 6x1011 vp/ kg of an earlier generation Ad vector was lethal in one of two partial OTC-deficient patients whereas a ½-log lower dose (2x1011 vp/kg) was well tolerated. Therefore, in considering clinical Ad-mediated liver-directed gene therapy, administration of lower doses is critical for safety. Unfortunately, while safe, systemic intravenous (IV) injection of lower doses is not efficacious because they yield little to no hepatocyte transduction. Therefore, it is important to develop novel strategies to increase the efficiency of hepatocyte transduction with low vector doses. Towards this goal, we have developed in nonhuman primates a minimally invasive method to achieve efficient hepatic transduction using low and safe HDAd doses. Briefly, a balloon catheter was percutaneously positioned in the inferior vena cava to occlude hepatic venous outflow and an HDAd expressing the baboon a-fetoprotein (bAFP) reporter was injected into the liver via a hepatic artery catheter and this method was compared to simple systemic peripheral IV injection. In these experiments, two baboons were injected with 3x1010 vp/kg HDAd by simple systemic peripheral IV injections (Fig. solid and open squares). As expected, at this low dose little to no transgene expression was detected. In contrast, when the same dose of 3x1010 vp/kg was administered to two baboons using the balloon occlusion catheter based approach (Fig. solid and open circles), an increased level of transgene expression of up to 80-fold was observed compared to systemic peripheral IV injection. In addition, transgene expression persisted at this high level for the duration of the observation period of at least 56 days. More importantly, when an even lower dose of 1x1010 vp/kg was administered to two baboons by the balloon occlusion catheter approach (Fig. solid and open triangles), the level of transgene expression was 30-fold higher than systemic peripheral IV injection of the higher 3x1010 vp/kg dose (Fig. solid and open squares). Thus, the balloon occlusion catheter technique permits delivery of clinically relevant low HDAd doses to achieve efficient hepatic transduction and may be a first step towards clinical application of HDAd for liver-directed gene therapy.
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