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513. Adenoviral Gene Transfer of Erythropoietin Confers Cytoprotection to Isolated Human Pancreatic Islets
NOVEL AD VECTORS AND APPLICATIONS (1E10iu). The luciferase activity (RLU/mg) measured 2 days after vector administration was similar between the two groups in the liver (5E5 for the 3 aggregated batches vs 6.4E5 for the 2 non aggregated batches), in the spleen and in the hart. In case of the secreted protein endostatin, the plasma levels achieved were also the same with aggregated virus (6.5mg/ml) as with non-aggregated virus (5.9mg/ml). Similar results were obtained with the ATF-BPTI transgene in Wag/Rij rats and in C57BL/6 mice. Additionally the transgene expression in muscle and liver were also not influenced by the presence of aggregation when Ad Luc (1E10 iu) was injected i.m. in C57BL/6 mice. The toxicity of two batches (n=6) of Ad Luc was evaluated, 3 days after iv administration, by histological examination of the liver, spleen, long, hart, bone, brain, kidney and limb muscle. The blind scoring of the damage included apoptosis, necrosis, mitosis, edema, vacuolar changes, and inflammation. As expected the non-aggregated batches induced mainly moderate liver toxicity (inflammation and vacuolar changes) and mild bone marrow damage and lung infiltration. The tissues of the aggregated group did not show any thrombi or ischemic or hemorrhagic lesions. Further the damage score of the different organs was similar in both groups. Thus it was concluded that the viral aggregation had no influence on the transgene expression and on the toxicity of recombinant adenovirus in cell culture as well as in rodents after iv administration.
512. Intra-Amniotic Application of CFTRExpressing Adenovirus Does Not Reverse Cystic Fibrosis Phenotype in Inbred Cftr-Knockout Mice Suzanne M. K. Buckley,1 Simon N. Waddington,1 Sarah Jezzard,1 Mike Themis,1 William H. Colledge,2 Charles Coutelle.1 1 Gene Therapy, Cell & Molecular Biology, Faculty of Medicine, Imperial College School of Medicine, London, United Kingdom; 2 Department of Physiology, University of Cambridge, Cambridge, United Kingdom. CF is characterized by early onset disease manifestation, which can already lead to perinatal intestinal complications such as meconium ileus. Pancreatic insufficiency and airway disease become manifest in early childhood, usually leading to severe and irreparable tissue damage which determines the final outcome of the disease. Because of early onset disease symptoms, cystic fibrosis was suggested to be a suitable disease for in utero gene therapy. In 1997, it was reported that the in utero administration at late gestation of a first generation adenovirus expressing CFTR, resulted in reversal of the cystic fibrosis phenotype in homozygote mice [Larson et al., 1997, Lancet, 349, 619-20]. This survival occurred despite the loss of detectable virus beyond 72 hours after its application and the lack of functional CFTR chloride channel activity in the jejunum of long-term surviving animals. This claim was met with considerable skepticism and criticism [Alton and Colledge, 1997, Lancet, 349, 1249-50]. Independent conformation of these findings is vital as this will determine whether CF gene therapy should be aimed at adults or fetuses. We therefore decided to reinvestigate this hypothesis using the same adenoviral vector and a null CF mouse line (CftrtmlCam) on a completely inbred genetic background (129Sv/ Ev) to remove the problem of genetic heterogeneity. After in vitro infection, RT-PCR and SPQ techniques were used to ensure that the virus construct expressed functional CFTR. Intra-amniotic injections were performed at day 16 gestation using a first-generation adenoviral vector expressing the human CFTR gene. Injected fetuses were harvested by caesarean section just before natural birth and fostered onto MF1 mothers to avoid cannibalization. Genotypes were determined at birth from umbilical cord samples and the survival of Cftr-/- mice was carefully monitored. We report that intra-amniotic injection with CFTR-expressing adenovirus did not result in any improvement of survival compared to untreated or mock-treated S200
animals. Therefore, our results do not support the claim that short term adenovirus-mediated expression of CFTR is sufficient to cure cystic fibrosis.
513. Adenoviral Gene Transfer of Erythropoietin Confers Cytoprotection to Isolated Human Pancreatic Islets Elizabeth S. Fenjves,1 Maria S. Ochoa,1 Carlota Gay-Rabinstein,1 Carlos A. Garcia,1 Armando J. Mendez,1 Camillo Ricordi.1 1 Diabetes Research Institute, University of Miami, School of Medicine, Miami, FL, United States. BACKGROUND: Type 1 diabetes is an autoimmune disease resulting in the destruction of the insulin-producing β cells of the islets of Langerhans. Pancreatic islet transplantation offers the potential to reestablish regulated insulin secretion in patients with this disease. Encouraging clinical results have been hampered by the fact that islets are functionally impaired in the transplant setting.Genetic engineering of islets with cytoprotective genes may prolong allograft survival and reduce the number of islets required for transplantation. Erythropoietin (EPO) is a kidney cytokine, which regulates haematopoiesis by promoting the survival, proliferation, and differentiation of erythroid progenitor cells. Recently, the role of EPO has been extended to other tissues where it appears to play a protective role by shielding against hypoxic, ischemic, apoptotic and necrotic stress. Furthermore, the EPO receptor has recently been reported by our laboratory to be expressed on pancreatic islets of various species. The goal of this research was to determine whether adenoviral-mediated gene transfer of EPO would have a cytoprotective effect on isolated human pancreatic islets. METHODS: Human pancreatic islets were isolated and transduced (500 pfu/cell) using either a recombinant, E1-deleted adenovirus carrying the human EPO gene and Green Fluorescent Protein (GFP) under the control of the cytomegalovirus promoter (AdCMVhEPO/GFP) or a similar virus coding only for GFP (AdCMVGFP). Transduction efficiency was measured by GFP fluorescence. Expression of EPO was measured after 12% SDSPAGE separation of 10ug of total proteins from infected and uninfected islets followed by electroblotting onto nitrocellulose membranes and immunodetection of human EPO with a monospecific polyclonal antibody. Apoptosis was induced either by serum starvation, overnight incubation in high glucose (166.5 mM) or long-term culture (2 weeks). Cells were analyzed for apoptosis measurement by flow cytometry for Annexin V-PE and differentiated from necrosis by PI. RESULTS: The average transduction efficiency of adenovirus in pancreatic islets was 65% as determined by fluorescence, and the average apoptosis level of untransduced untreated islets was 50%. Western blot analysis confirmed EPO expression in the islets transduced with EPO but not in control islets. EPO transduced islets were significantly protected from apoptosis. In the case of induced apoptosis protection levels were between 5 and 10 fold. In the case of spontaneous apoptosis in long-term culture the protection was 2 fold. DISCUSSION: Transduction with adenovirus coding for EPO significantly reduced apoptosis in human islets under various conditions. EPO may be useful in the engineering of pancreatic islets to improve survival in the transplant setting.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
512. Intra-Amniotic Application of CFTRExpressing Adenovirus Does Not Reverse Cystic Fibrosis Phenotype in Inbred Cftr-Knockout Mice Suzanne M. K. Buckley,1 Simon N. Waddington,1 Sarah Jezzard,1 Mike Themis,1 William H. Colledge,2 Charles Coutelle.1 1 Gene Therapy, Cell & Molecular Biology, Faculty of Medicine, Imperial College School of Medicine, London, United Kingdom; 2 Department of Physiology, University of Cambridge, Cambridge, United Kingdom. CF is characterized by early onset disease manifestation, which can already lead to perinatal intestinal complications such as meconium ileus. Pancreatic insufficiency and airway disease become manifest in early childhood, usually leading to severe and irreparable tissue damage which determines the final outcome of the disease. Because of early onset disease symptoms, cystic fibrosis was suggested to be a suitable disease for in utero gene therapy. In 1997, it was reported that the in utero administration at late gestation of a first generation adenovirus expressing CFTR, resulted in reversal of the cystic fibrosis phenotype in homozygote mice [Larson et al., 1997, Lancet, 349, 619-20]. This survival occurred despite the loss of detectable virus beyond 72 hours after its application and the lack of functional CFTR chloride channel activity in the jejunum of long-term surviving animals. This claim was met with considerable skepticism and criticism [Alton and Colledge, 1997, Lancet, 349, 1249-50]. Independent conformation of these findings is vital as this will determine whether CF gene therapy should be aimed at adults or fetuses. We therefore decided to reinvestigate this hypothesis using the same adenoviral vector and a null CF mouse line (CftrtmlCam) on a completely inbred genetic background (129Sv/ Ev) to remove the problem of genetic heterogeneity. After in vitro infection, RT-PCR and SPQ techniques were used to ensure that the virus construct expressed functional CFTR. Intra-amniotic injections were performed at day 16 gestation using a first-generation adenoviral vector expressing the human CFTR gene. Injected fetuses were harvested by caesarean section just before natural birth and fostered onto MF1 mothers to avoid cannibalization. Genotypes were determined at birth from umbilical cord samples and the survival of Cftr-/- mice was carefully monitored. We report that intra-amniotic injection with CFTR-expressing adenovirus did not result in any improvement of survival compared to untreated or mock-treated S200
animals. Therefore, our results do not support the claim that short term adenovirus-mediated expression of CFTR is sufficient to cure cystic fibrosis.
513. Adenoviral Gene Transfer of Erythropoietin Confers Cytoprotection to Isolated Human Pancreatic Islets Elizabeth S. Fenjves,1 Maria S. Ochoa,1 Carlota Gay-Rabinstein,1 Carlos A. Garcia,1 Armando J. Mendez,1 Camillo Ricordi.1 1 Diabetes Research Institute, University of Miami, School of Medicine, Miami, FL, United States. BACKGROUND: Type 1 diabetes is an autoimmune disease resulting in the destruction of the insulin-producing β cells of the islets of Langerhans. Pancreatic islet transplantation offers the potential to reestablish regulated insulin secretion in patients with this disease. Encouraging clinical results have been hampered by the fact that islets are functionally impaired in the transplant setting.Genetic engineering of islets with cytoprotective genes may prolong allograft survival and reduce the number of islets required for transplantation. Erythropoietin (EPO) is a kidney cytokine, which regulates haematopoiesis by promoting the survival, proliferation, and differentiation of erythroid progenitor cells. Recently, the role of EPO has been extended to other tissues where it appears to play a protective role by shielding against hypoxic, ischemic, apoptotic and necrotic stress. Furthermore, the EPO receptor has recently been reported by our laboratory to be expressed on pancreatic islets of various species. The goal of this research was to determine whether adenoviral-mediated gene transfer of EPO would have a cytoprotective effect on isolated human pancreatic islets. METHODS: Human pancreatic islets were isolated and transduced (500 pfu/cell) using either a recombinant, E1-deleted adenovirus carrying the human EPO gene and Green Fluorescent Protein (GFP) under the control of the cytomegalovirus promoter (AdCMVhEPO/GFP) or a similar virus coding only for GFP (AdCMVGFP). Transduction efficiency was measured by GFP fluorescence. Expression of EPO was measured after 12% SDSPAGE separation of 10ug of total proteins from infected and uninfected islets followed by electroblotting onto nitrocellulose membranes and immunodetection of human EPO with a monospecific polyclonal antibody. Apoptosis was induced either by serum starvation, overnight incubation in high glucose (166.5 mM) or long-term culture (2 weeks). Cells were analyzed for apoptosis measurement by flow cytometry for Annexin V-PE and differentiated from necrosis by PI. RESULTS: The average transduction efficiency of adenovirus in pancreatic islets was 65% as determined by fluorescence, and the average apoptosis level of untransduced untreated islets was 50%. Western blot analysis confirmed EPO expression in the islets transduced with EPO but not in control islets. EPO transduced islets were significantly protected from apoptosis. In the case of induced apoptosis protection levels were between 5 and 10 fold. In the case of spontaneous apoptosis in long-term culture the protection was 2 fold. DISCUSSION: Transduction with adenovirus coding for EPO significantly reduced apoptosis in human islets under various conditions. EPO may be useful in the engineering of pancreatic islets to improve survival in the transplant setting.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy