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1087. Balloon Occlusion Catheter-Based Delivery of HDAd into the Nonhuman Primate Liver Results in Stable, High Level Transgene Expression with Minimal Toxicity
IMMUNODEFICIENCIES/HEMOPHILIA IMMUNODEFICIENCIES/HEMOPHILIA 1086. T Cell Responses to Canine Factor IX and AAV Capsid Antigens in Hemophilia B Dogs after Intravascular Gene Delivery to Skeletal Muscle 1
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Daniel J. Hui, Federico Mingozzi, Aaron Dillow, Stephanie McCorquodale,2 Timothy C. Nichols,2 Valder R. Arruda,1 Katherine A. High.1 1 Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA; 2Pathology, University of North Carolina, Chapel Hill, Chapel Hill, NC. Previous studies have shown successful correction of hemophilia B in dogs by muscle-directed gene delivery of canine F.IX (cF.IX) using an adeno-associated virus (AAV) serotype 2. Intravascular delivery of the cF.IX gene to skeletal muscle allowed long-term expression of cF.IX of 4-20%, thereby correcting the disease phenotype. However, concerns over the risk of immune responses against the transgene and viral vector antigens have focused recent work on further characterizing the immunogenicity of both AAV capsid and F.IX in a gene therapy setting. Six HB dogs from the Chapel Hill colony received three different doses of an AAV2-cF.IX vector (1x1012 vg/kg, n=3; 3 x 1012 vg/kg, n=2; 8 x 1012 vg/kg, n=1) by intravascular delivery to skeletal muscle, in addition to an immunosuppression (IS) regimen (6 weekly infusions of cyclophosphamide beginning at the time of vector administration). T cell responses were measured on PBMCs isolated from whole blood drawn prior to vector infusion, during IS and after discontinuing IS. PBMCs were used for ELISpot assays to measure IL-10 and IFN-γ secretion in response to both cF.IX and AAV-2 antigens. This approach utilized a peptide library consisting of 15mers overlapping by 10 amino acids, spanning the entire antigen sequence, arranged in a matrix such that each peptide was contained in two orthogonal pools. All HB dogs in this study exhibited a unique Th2 response consisting of IL-10 secretion in response to a common epitope corresponding to peptide 68 in the cF.IX library. This epitope was also found in normal dogs, which have the natural form of the protein, suggesting a role in tolerance to the cF.IX transgene rather than immunity. Interestingly, the dog that received the highest vector dose (1.5 µg cF.IX/ml, 30%) at day 15 postinjection, developed a non-neutralizing antibody response to cF.IX, as well as a T cell response to an epitope corresponding to peptide 84. This peptide maps to the region containing the missense mutation responsible for the disease phenotype (Chapel Hill mutation, Glu379→Gly). No IFN-γ secretion was detected following AAV2-cF.IX gene transfer to muscle, contrasting the Th1 response previously seen in intramuscularly delivered cF.IX using an AAV-1 vector. While all animals that received an AAV2-cF.IX vector developed B cell responses against the viral capsid antigen, T cell responses to the capsid were undetectable in PBMCs even after in vitro expansion with the antigen. In conclusion, intravascular delivery of AAV2-cF.IX to skeletal muscle elicited a Th2 T cell response characterized by the secretion of IL-10 in response to a specific cF.IX peptide. An additional immunodominant peptide was detected in the presence of a nonneutralizing immune response to the cF.IX transgene at the highest dose. These results suggest that while route of administration and/ or AAV serotype influence the type of immune response elicited, transgene level may impose additional safety limitations in terms of T cell response.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
1087. Balloon Occlusion Catheter-Based Delivery of HDAd into the Nonhuman Primate Liver Results in Stable, High Level Transgene Expression with Minimal Toxicity Nicola Brunetti-Pierri,1 Gary Stapleton,2 Donna Palmer,1 Yu Zuo,1 Arthur Beaudet,1 Charles Mullins,2 Philip Ng.1 1 Molecular and Human 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 because they can mediate long-term transgene expression without chronic toxicity. However, due to a nonlinear dose-response, high doses are required to achieve efficient hepatic transduction resulting in dose-dependent acute toxicity. To overcome this important obstacle, we have developed a minimally invasive method to preferentially deliver low dose HDAd into the liver of 30 kg baboons to achieve efficient hepatic transduction. Briefly, a single sausage-shaped balloon occlusion catheter was percutaneously positioned in the inferior vena cava (IVC) of baboon 1 to occlude hepatic venous outflow (Fig. A). 1x1011 vp/kg of a HDAd expressing the baboon alpha-fetoprotein (bAFP) marker was injected directly into the occluded liver via a percutaneously placed hepatic artery (HA) catheter and left to dwell within the liver for 15 min before balloon deflation. As controls, 1x1011 vp/kg was administered to baboon 2 by peripheral intravenous injection and baboon 3 by HA injection without balloon occlusion. All procedures were uneventful, well tolerated, and following recovery from anesthesia, all three animals returned to their normal, active pre-injection states with no clinical manifestations of toxicity. Mild transaminitis was observed for all animals, peaking at 24 to 48 h post-vector but returning towards baseline the next day: For ALT, a 2.3, 1.1, and 1.4-fold increase over baseline were observed for baboons 1, 2 and 3, respectively. For AST, a 4.8, 1.6 and 2.8-fold increase were observed for baboons 1, 2 and 3, respectively. Importantly, serum bAFP levels increased 425-fold over baseline for baboon 1 (from 4.5 ng/ml at a baseline to 1914 ng/ml), while only a modest increase of 12-fold and 5.6-fold were observed for baboon 2 (from 9 ng/ml to 109 ng/ml) and baboon 3 (from 18 ng/ml to 103 ng/ml), respectively (Fig. B). To date, these bAFP levels have been sustained (at least 56 days). These results suggest that the therapeutic index of HDAd can be significantly improved by delivering the vector preferentially into the liver using a minimally invasive balloon occlusion catheter technique and may be a first step towards clinical application of HDAd for liver-directed gene therapy.
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Daniel J. Hui, Federico Mingozzi, Aaron Dillow, Stephanie McCorquodale,2 Timothy C. Nichols,2 Valder R. Arruda,1 Katherine A. High.1 1 Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA; 2Pathology, University of North Carolina, Chapel Hill, Chapel Hill, NC. Previous studies have shown successful correction of hemophilia B in dogs by muscle-directed gene delivery of canine F.IX (cF.IX) using an adeno-associated virus (AAV) serotype 2. Intravascular delivery of the cF.IX gene to skeletal muscle allowed long-term expression of cF.IX of 4-20%, thereby correcting the disease phenotype. However, concerns over the risk of immune responses against the transgene and viral vector antigens have focused recent work on further characterizing the immunogenicity of both AAV capsid and F.IX in a gene therapy setting. Six HB dogs from the Chapel Hill colony received three different doses of an AAV2-cF.IX vector (1x1012 vg/kg, n=3; 3 x 1012 vg/kg, n=2; 8 x 1012 vg/kg, n=1) by intravascular delivery to skeletal muscle, in addition to an immunosuppression (IS) regimen (6 weekly infusions of cyclophosphamide beginning at the time of vector administration). T cell responses were measured on PBMCs isolated from whole blood drawn prior to vector infusion, during IS and after discontinuing IS. PBMCs were used for ELISpot assays to measure IL-10 and IFN-γ secretion in response to both cF.IX and AAV-2 antigens. This approach utilized a peptide library consisting of 15mers overlapping by 10 amino acids, spanning the entire antigen sequence, arranged in a matrix such that each peptide was contained in two orthogonal pools. All HB dogs in this study exhibited a unique Th2 response consisting of IL-10 secretion in response to a common epitope corresponding to peptide 68 in the cF.IX library. This epitope was also found in normal dogs, which have the natural form of the protein, suggesting a role in tolerance to the cF.IX transgene rather than immunity. Interestingly, the dog that received the highest vector dose (1.5 µg cF.IX/ml, 30%) at day 15 postinjection, developed a non-neutralizing antibody response to cF.IX, as well as a T cell response to an epitope corresponding to peptide 84. This peptide maps to the region containing the missense mutation responsible for the disease phenotype (Chapel Hill mutation, Glu379→Gly). No IFN-γ secretion was detected following AAV2-cF.IX gene transfer to muscle, contrasting the Th1 response previously seen in intramuscularly delivered cF.IX using an AAV-1 vector. While all animals that received an AAV2-cF.IX vector developed B cell responses against the viral capsid antigen, T cell responses to the capsid were undetectable in PBMCs even after in vitro expansion with the antigen. In conclusion, intravascular delivery of AAV2-cF.IX to skeletal muscle elicited a Th2 T cell response characterized by the secretion of IL-10 in response to a specific cF.IX peptide. An additional immunodominant peptide was detected in the presence of a nonneutralizing immune response to the cF.IX transgene at the highest dose. These results suggest that while route of administration and/ or AAV serotype influence the type of immune response elicited, transgene level may impose additional safety limitations in terms of T cell response.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
1087. Balloon Occlusion Catheter-Based Delivery of HDAd into the Nonhuman Primate Liver Results in Stable, High Level Transgene Expression with Minimal Toxicity Nicola Brunetti-Pierri,1 Gary Stapleton,2 Donna Palmer,1 Yu Zuo,1 Arthur Beaudet,1 Charles Mullins,2 Philip Ng.1 1 Molecular and Human 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 because they can mediate long-term transgene expression without chronic toxicity. However, due to a nonlinear dose-response, high doses are required to achieve efficient hepatic transduction resulting in dose-dependent acute toxicity. To overcome this important obstacle, we have developed a minimally invasive method to preferentially deliver low dose HDAd into the liver of 30 kg baboons to achieve efficient hepatic transduction. Briefly, a single sausage-shaped balloon occlusion catheter was percutaneously positioned in the inferior vena cava (IVC) of baboon 1 to occlude hepatic venous outflow (Fig. A). 1x1011 vp/kg of a HDAd expressing the baboon alpha-fetoprotein (bAFP) marker was injected directly into the occluded liver via a percutaneously placed hepatic artery (HA) catheter and left to dwell within the liver for 15 min before balloon deflation. As controls, 1x1011 vp/kg was administered to baboon 2 by peripheral intravenous injection and baboon 3 by HA injection without balloon occlusion. All procedures were uneventful, well tolerated, and following recovery from anesthesia, all three animals returned to their normal, active pre-injection states with no clinical manifestations of toxicity. Mild transaminitis was observed for all animals, peaking at 24 to 48 h post-vector but returning towards baseline the next day: For ALT, a 2.3, 1.1, and 1.4-fold increase over baseline were observed for baboons 1, 2 and 3, respectively. For AST, a 4.8, 1.6 and 2.8-fold increase were observed for baboons 1, 2 and 3, respectively. Importantly, serum bAFP levels increased 425-fold over baseline for baboon 1 (from 4.5 ng/ml at a baseline to 1914 ng/ml), while only a modest increase of 12-fold and 5.6-fold were observed for baboon 2 (from 9 ng/ml to 109 ng/ml) and baboon 3 (from 18 ng/ml to 103 ng/ml), respectively (Fig. B). To date, these bAFP levels have been sustained (at least 56 days). These results suggest that the therapeutic index of HDAd can be significantly improved by delivering the vector preferentially into the liver using a minimally invasive balloon occlusion catheter technique and may be a first step towards clinical application of HDAd for liver-directed gene therapy.
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