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1180. Complete, Long-Term Correction of Hyperbilirubinemia in the Gunn Rat Model of Crigler-Najjar Syndrome
NEW APPROACHES FOR CORRECTION OF GENETIC AND METABOLIC DISEASES genome to reflect a hepatocyte expression profile. In conclusion, fusion of donor-derived hematopoietic cells with host hepatocytes can correct the liver function in a metabolic disorder, and may be effective in treating other diseases as well. Furthermore, our results suggest that cell fusion could underlie other examples of presumed stem cell plasticity.
1179. Retroviral Marking of Bone Marrow Derived Hepatocytes Patrice K. Held,1 Meenakshi Noll,1 Eugenio Montini,1 Francesco Galimi,2 Muhsen Al-Dhalimy,1 Milton Finegold,3 Inder Verma,2 Markus Grompe.1 1 Department of Molecular and Medical Genetics, Oregon Health & Sciences University, Portland, OR; 2Laboratory of Genetics, Salk Institute, La Jolla, CA; 3Department of Pathology, Texas Children’s Hospital, Houston, TX. Hematopoietic stem cell (HSC) transplantation results in liver repopulation in the murine model of Hereditary Tyrosinemia Type I (HTI), deficient in fumarylacetoacetate hydrolase (Fah). In addition, all hematopoietic lineages are also replaced by the transplanted donor cells. However, it was unknown whether the same clonal precursor contributed to both liver and blood reconstitution, or whether different stem cells were used. In order to examine the clonality of liver and blood repopulation, retroviral marking of bone marrow cells was used. In two independent experiments, both lentiviral and oncoretroviral vectors were used. Bone marrow was harvested from a donor Fah-/- mouse and transduced with a viral vector carrying human Fah cDNA. For the lentiviral transduction, an overnight incubation was used. For the oncoretroviral vector, Fah mutant marrow and packaging cells were co-cultured for 3 days. The infected bone marrow was then transplanted retro-orbitally into lethally irradiated Fah-/- recipient mice. After hematopoietic repopulation, the recipient mice were removed from the liver protective drug, NTBC, to allow for liver repopulation. After 90 days of selection for Fah+ hepatocytes (approximately 60% repopulation), the liver and hematopoietic tissues were harvested. The viral integration sites were analyzed by Southern blot using multiple different restriction enzymes, which cut once in the provirus. Multiple retroviral integrations sites were detectable in all hematopoietic lineages. In contrast, hepatocyte DNA showed only a single integration site in the two independent animals analyzed to date. This site was identical to one of the sites found in hematopoietic tissues, thus proving clonality. Blood cells and hepatocytes did indeed arise from the same HSC and the existence of an independent bone marrow resident hepatocyte stem cell could be ruled out. The marking data are consistent with our recent finding that bone marrow derived hepatocytes arise by cell fusion between retrovirally marked hematopoietic cells and host hepatocytes. Two alternative explanations exist for the presence of only one integration site in hepatocytes. First, it is possible that only a subset of HSC contributes to cell fusion in the liver (“fusogenic stem cell”). Second, and more likely, only a subset integration sites may result in levels of Fah expression sufficient to achieve hepatocyte selection. These experiments also serve as a proof of principle that retroviruses can be used to correct HSC and cure liver disease associated with HTI.
1180. Complete, Long-Term Correction of Hyperbilirubinemia in the Gunn Rat Model of Crigler-Najjar Syndrome Gabriele Toietta,1 Viraj P. Mane,1 Brendan Lee,1,2 Arthur L. Beaudet.1 1 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States; 2Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX, United States. Crigler-Najjar syndrome type I is a rare inborn error of bilirubin metabolism due to a deficiency of the hepatic microsomal bilirubin uridine diphosphate-glucuronosyltransferase (B-UDPGT) enzyme. To reduce the risk of life threatening kernicterus, patients undergo daily session of phototherapy. The only therapeutic alternatives are liver or hepatocyte transplantation but significant risks and costs are associated with these procedures. Since all other pathways for bilirubin metabolism are functional in the patients, gene therapy may represent an alternative treatment. An excellent model of this disease exists in the Gunn rat. Previously, partial correction of hyperbilirubinemia in Gunn rats for up to 4 weeks was reported using E1-deleted adenoviral (Ad) vectors; however, the therapeutic index of E1-deleted Ad is limited due to strong host immune response to the viral proteins, acute toxicity and short duration of expression. Helper-dependent adenoviral (HD-Ad) vectors, devoid of all viral coding sequences, have a safer chronic toxicity profile compared with early generation Ad vectors and allow for long-term transgene expression. We used HD-Ad vectors and tissue-restricted expression of human B-UDPGT in Gunn rats. Complete correction of hyperbilirubinemia was achieved after a single systemic administration of 3 x 1012 viral particles/kg of HD-Ad vectors expressing human B-UDPGT under the control of the rat phosphoenolpyruvate carboxylase (PEPCK) promoter into Gunn rats. Total bilirubin levels in the plasma were stably reduced from above 7.0 mg/dL to less than 1.0 mg/dL for at least 7 months. Specific hepatic expression of human B-UDPGT was confirmed by RNA and protein analysis in liver biopsy from animals sacrificed 20 weeks post-injection. A clinically significant reduction in total bilirubin levels was observed with a dose as low as 6 x 1011 viral particles/kg. No significant increase in liver enzymes was detected at a dose as high as 1 x 10 13 viral particles/kg. Transient, dose-related thrombocytopenia developed after systemic injection of the vector at higher doses and resolved in 3 weeks. We conclude that complete, long-term correction of hyperbilirubinemia may be achieved by a single HD-Ad adenoviral administration in the animal model of Crigler-Najjar syndrome type I with negligible chronic toxicity.
1181. Acute Toxicity Following High Dose Systemic Delivery of Helper-Dependent Adenoviral Vectors into Nonhuman Primates Philip Ng,1 Nicola Brunetti-Pierri,1 Donna Palmer,1 Dee Carey,2 Milton Finegold,3 Arthur Beaudet.1 1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2Southwest Foundation for Biomedical Research, San Antonio, TX; 3Department of Pathology, Baylor College of Medicine, Houston, TX. Systemic delivery by intravascular injection of adenoviral (Ad) vectors for liver-directed gene transfer has been a widely employed gene therapy strategy due to its simplicity and noninvasiveness. In the case of early generation Ad vectors, this results in transient high level transgene expression and long-term chronic hepatotoxicity due to viral gene expression from the vector backbone. Furthermore, systemic delivery of high doses results in an acute innate inflammatory response with severe and lethal consequences. Unlike early generation Ad vectors, helper-dependent Ad vectors (HDAd) contain no viral coding sequences and can thus provide prolonged
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Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy
1179. Retroviral Marking of Bone Marrow Derived Hepatocytes Patrice K. Held,1 Meenakshi Noll,1 Eugenio Montini,1 Francesco Galimi,2 Muhsen Al-Dhalimy,1 Milton Finegold,3 Inder Verma,2 Markus Grompe.1 1 Department of Molecular and Medical Genetics, Oregon Health & Sciences University, Portland, OR; 2Laboratory of Genetics, Salk Institute, La Jolla, CA; 3Department of Pathology, Texas Children’s Hospital, Houston, TX. Hematopoietic stem cell (HSC) transplantation results in liver repopulation in the murine model of Hereditary Tyrosinemia Type I (HTI), deficient in fumarylacetoacetate hydrolase (Fah). In addition, all hematopoietic lineages are also replaced by the transplanted donor cells. However, it was unknown whether the same clonal precursor contributed to both liver and blood reconstitution, or whether different stem cells were used. In order to examine the clonality of liver and blood repopulation, retroviral marking of bone marrow cells was used. In two independent experiments, both lentiviral and oncoretroviral vectors were used. Bone marrow was harvested from a donor Fah-/- mouse and transduced with a viral vector carrying human Fah cDNA. For the lentiviral transduction, an overnight incubation was used. For the oncoretroviral vector, Fah mutant marrow and packaging cells were co-cultured for 3 days. The infected bone marrow was then transplanted retro-orbitally into lethally irradiated Fah-/- recipient mice. After hematopoietic repopulation, the recipient mice were removed from the liver protective drug, NTBC, to allow for liver repopulation. After 90 days of selection for Fah+ hepatocytes (approximately 60% repopulation), the liver and hematopoietic tissues were harvested. The viral integration sites were analyzed by Southern blot using multiple different restriction enzymes, which cut once in the provirus. Multiple retroviral integrations sites were detectable in all hematopoietic lineages. In contrast, hepatocyte DNA showed only a single integration site in the two independent animals analyzed to date. This site was identical to one of the sites found in hematopoietic tissues, thus proving clonality. Blood cells and hepatocytes did indeed arise from the same HSC and the existence of an independent bone marrow resident hepatocyte stem cell could be ruled out. The marking data are consistent with our recent finding that bone marrow derived hepatocytes arise by cell fusion between retrovirally marked hematopoietic cells and host hepatocytes. Two alternative explanations exist for the presence of only one integration site in hepatocytes. First, it is possible that only a subset of HSC contributes to cell fusion in the liver (“fusogenic stem cell”). Second, and more likely, only a subset integration sites may result in levels of Fah expression sufficient to achieve hepatocyte selection. These experiments also serve as a proof of principle that retroviruses can be used to correct HSC and cure liver disease associated with HTI.
1180. Complete, Long-Term Correction of Hyperbilirubinemia in the Gunn Rat Model of Crigler-Najjar Syndrome Gabriele Toietta,1 Viraj P. Mane,1 Brendan Lee,1,2 Arthur L. Beaudet.1 1 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States; 2Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX, United States. Crigler-Najjar syndrome type I is a rare inborn error of bilirubin metabolism due to a deficiency of the hepatic microsomal bilirubin uridine diphosphate-glucuronosyltransferase (B-UDPGT) enzyme. To reduce the risk of life threatening kernicterus, patients undergo daily session of phototherapy. The only therapeutic alternatives are liver or hepatocyte transplantation but significant risks and costs are associated with these procedures. Since all other pathways for bilirubin metabolism are functional in the patients, gene therapy may represent an alternative treatment. An excellent model of this disease exists in the Gunn rat. Previously, partial correction of hyperbilirubinemia in Gunn rats for up to 4 weeks was reported using E1-deleted adenoviral (Ad) vectors; however, the therapeutic index of E1-deleted Ad is limited due to strong host immune response to the viral proteins, acute toxicity and short duration of expression. Helper-dependent adenoviral (HD-Ad) vectors, devoid of all viral coding sequences, have a safer chronic toxicity profile compared with early generation Ad vectors and allow for long-term transgene expression. We used HD-Ad vectors and tissue-restricted expression of human B-UDPGT in Gunn rats. Complete correction of hyperbilirubinemia was achieved after a single systemic administration of 3 x 1012 viral particles/kg of HD-Ad vectors expressing human B-UDPGT under the control of the rat phosphoenolpyruvate carboxylase (PEPCK) promoter into Gunn rats. Total bilirubin levels in the plasma were stably reduced from above 7.0 mg/dL to less than 1.0 mg/dL for at least 7 months. Specific hepatic expression of human B-UDPGT was confirmed by RNA and protein analysis in liver biopsy from animals sacrificed 20 weeks post-injection. A clinically significant reduction in total bilirubin levels was observed with a dose as low as 6 x 1011 viral particles/kg. No significant increase in liver enzymes was detected at a dose as high as 1 x 10 13 viral particles/kg. Transient, dose-related thrombocytopenia developed after systemic injection of the vector at higher doses and resolved in 3 weeks. We conclude that complete, long-term correction of hyperbilirubinemia may be achieved by a single HD-Ad adenoviral administration in the animal model of Crigler-Najjar syndrome type I with negligible chronic toxicity.
1181. Acute Toxicity Following High Dose Systemic Delivery of Helper-Dependent Adenoviral Vectors into Nonhuman Primates Philip Ng,1 Nicola Brunetti-Pierri,1 Donna Palmer,1 Dee Carey,2 Milton Finegold,3 Arthur Beaudet.1 1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2Southwest Foundation for Biomedical Research, San Antonio, TX; 3Department of Pathology, Baylor College of Medicine, Houston, TX. Systemic delivery by intravascular injection of adenoviral (Ad) vectors for liver-directed gene transfer has been a widely employed gene therapy strategy due to its simplicity and noninvasiveness. In the case of early generation Ad vectors, this results in transient high level transgene expression and long-term chronic hepatotoxicity due to viral gene expression from the vector backbone. Furthermore, systemic delivery of high doses results in an acute innate inflammatory response with severe and lethal consequences. Unlike early generation Ad vectors, helper-dependent Ad vectors (HDAd) contain no viral coding sequences and can thus provide prolonged
S456
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy