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1009. Gene Silencing through RNA Interference and Antisense Strategies in the Lung
GENETIC AND METABOLIC DISEASES: PART TWO 60 and TRA-1-81 is in progress. Multipotency of ES-like cells will be determined by the generation of teratomas consisting of cell derivatives of the three embryonic germ cell layers after injection of cells into SCID mice. This work was supported by research grants from the Canadian Institutes of Health Research and Network Centre of Excellence, Stem Cell Network.
1009. Gene Silencing through RNA Interference and Antisense Strategies in the Lung Uta Griesenbach,1,7 Chris Kitson,2 Sara Escudero Garcia,1,7 Hazel Painter,3,7 Raymond Farley,1,7 Yu-Hua Chow,4 Mark Edbrooke,2 Seng Cheng,5 Jim Hu,4 Steve Hyde,3,7 Duncan Geddes,1,7 Natasha Caplen,6 Eric Alton. 1 Department of Gene Therapy, Faculty of Medicine, Imperial College London, London, United Kingdom; 2GlaxoSmithKline, Stevenage, United Kingdom; 3Gene Medicine Group, Nuffield Department of Clinical Laboratory Science, University of Oxford, Oxford, United Kingdom; 4Hospital for Sich Children, Toronto, Canada; 5Genzyme Coporation, Framingham, United States; 6 Medical Genetics Branch, NHGRI, NIH, Bethesda, MA, United States; 7UK Cystic Fibrosis Gene Therapy Consortium, United Kingdom. Increased sodium absorption across the airway epithelium is a characteristic feature of cystic fibrosis (CF) lung disease and is likely to contribute significantly to disease aetiology. We are developing a programme aimed at decreasing the activity of the epithelial sodium channel (ENaC) in airway epithelial cells in vivo using antisense (AS) strategies and RNA interference (RNAi) based on transfection of single stranded DNA (ssDNA) oligomers and small interfering RNAs (siRNAs), respectively. We are targeting βgalactosidase (βgal) in K18-lacZ transgenic mice as a surrogate protein. These animals express βgal under the control of the cytokeratin 18 (K18) promoter, which has a very similar expression profile to the CF gene in airway epithelial cells. First, ssDNA and siRNAs molecules against the αENaC subunit were assessed in murine kidney cells (M1), which express high levels of ENaC and are suitable for electrophysiological analysis of ENaC activity. Ss DNA and siRNAs against βgal were assessed in NIH-3T3-lacZ cells, which stably express βgal. We designed 5 ENaCand 5 lacZssDNA (20 mers), using computer sided design. Two out of 5 ENaC AS molecules reduced mRNA expression by approximately 60% (n=6, p<0.05) and 2 out of five lacZAS reduced mRNA expression by about 70% (n=6, p<0.05) in a dose-dependent fashion. We assessed 3 ENaC siRNA molecules (none of which has been effective in reducing ENaC mRNA levels) and 10 lacZ siRNAs, of which three were effective in reducing βgal expression by at least 50% (p<0.05). The rational design of siRNA for RNAi is not well understood and emperical changes may necessary to identify the most effective molecules. We also transfected M1 cells with FITClabelled ssDNA and siRNAs and analysed intracellular distribution at different time-points after transfection (1,15,30,60min and 2,4,6,8 and 24 hrs). Although transfection efficiency was similar the intracellular distribution of these molecules was very different at all time-points. The ssDNA was visible as diffuse staining as early as 30 min after transfection and preferentially accumulated in the nuclei at all time-points, whereas the siRNAs was not visible in the nucleus at any time-point, but accumulated in the cytoplasm of transfected cells. This intracellular distribution of ssDNA and siRNA is consistent with the presumed site of action for antisense and RNAi, respectively. We have begun in vivo studies with lacZ siRNA. The lungs of K18-lacZ mice were transfected with lipid (GL67)-complexed lacZsiRNA via a standard “sniffing” protocol. Forty-eight hrs after transfection lacZ mRNA was reduced 30%, when compared to control animals (n=14, p<0.01). Interestingly, in vivo bio-distribution Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
studies using FITC-dsRNA showed no signal in airway epithelial cells 1 or 24 hrs after transfection. This may be due to degradation, with only small amounts of dsRNA being taken up into the cell. Further optimisation of RNAi and studies using antisense molecules in the lung are currently under way.
1010. Adenoviral and AAV Vectors for an Efficient Intracerebral Gene Transfer in the Sandhoff Mouse Model Christophe A. Bourgoin,1 Carla Emiliani,2 Antoinette Gelot,3 Brunela Tancini,2 Montanucci Pia,2 Livia Poenaru,1 Catherine Caillaud.1 1 Departement GDPM, Institut Cochin (INSERM, CNRS, Universite Paris 5), Paris, France; 2Biochemical Sciences and Molecular Biotechnologies Department, University of Perugia, Perugia, Italy; 3Unite de Neuropathologie, Hopital Trousseau, Paris, France. Sandhoff disease is an autosomal recessive neurodegenerative disorder characterized by the intralysosomal accumulation of GM2 ganglioside. It is due to mutations in the HEXB gene encoding the hexosaminidases β-chain and results in a hexosaminidases A (αβ) and B (ββ) deficiency. In order to test the feasibility of gene transfer methods in this model, a recombinant adenoviral vector encoding the β-subunit was injected intracerebrally into the murine model of Sandhoff disease. It leaded to a normal hexosaminidases total activity in the entire brain and a partial restoration of the Hex A specific activity. The coadministration of hyperosmotic concentrations of mannitol with the adenoviral vector resulted in a drastic enhancement of the vector diffusion in the injected hemisphere and significantly improved transduction efficiency. Our data underline the advantage of the use of mannitol in combination with low doses of adenoviral vectors, allowing a high and widespread expression without any adeno-related lesions. AAV vectors containing the human HEXA and HEXB cDNAs under the control of the CAG promoter were also constructed. AAV-HEXB was first injected alone into the brain of hexb -/neonates. Histological staining showed a high enzymatic activity in widely diffuse areas. Hexosaminidases activity was restored to the normal level in the whole brain, but Hex A levels remained low. The coadministration of both HEXA and HEXB vectors in the CNS permitted to reach therapeutic levels of hexosaminidases A and B, as demonstrated by the GM2 ganglioside degradation, confirming the necessity of supplying both subunits in the case of heterodimeric proteins, such as Hex A.
1011. Gastric Acid Neutralization and Protease Inhibition Improves Oral Recombinant AdenoAssociated Virus-Mediated Gene Delivery to the Intestines Guohong Shao,1 Kristin C. Backstrom,1 Qin Huang,1 Thomas J. Sferra.1 1 Center for Gene Therapy, Children’s Research Institute, Columbus, OH. Gene transfer to the gastrointestinal tract mediated by recombinant adeno-associated virus (rAAV) vectors has many potential applications including complementation of single gene disorders, genetic immunization and production of therapeutic molecules to act at local or distant sites. In vitro studies indicate that rAAV serotype 2 vectors are adversely affected by conditions within the GI tract (e.g. low intragastric pH, secreted proteases). The objective of this study was to evaluate whether acid neutralization and protease inhibition can improve rAAV gene delivery to the intestines. S389
1009. Gene Silencing through RNA Interference and Antisense Strategies in the Lung Uta Griesenbach,1,7 Chris Kitson,2 Sara Escudero Garcia,1,7 Hazel Painter,3,7 Raymond Farley,1,7 Yu-Hua Chow,4 Mark Edbrooke,2 Seng Cheng,5 Jim Hu,4 Steve Hyde,3,7 Duncan Geddes,1,7 Natasha Caplen,6 Eric Alton. 1 Department of Gene Therapy, Faculty of Medicine, Imperial College London, London, United Kingdom; 2GlaxoSmithKline, Stevenage, United Kingdom; 3Gene Medicine Group, Nuffield Department of Clinical Laboratory Science, University of Oxford, Oxford, United Kingdom; 4Hospital for Sich Children, Toronto, Canada; 5Genzyme Coporation, Framingham, United States; 6 Medical Genetics Branch, NHGRI, NIH, Bethesda, MA, United States; 7UK Cystic Fibrosis Gene Therapy Consortium, United Kingdom. Increased sodium absorption across the airway epithelium is a characteristic feature of cystic fibrosis (CF) lung disease and is likely to contribute significantly to disease aetiology. We are developing a programme aimed at decreasing the activity of the epithelial sodium channel (ENaC) in airway epithelial cells in vivo using antisense (AS) strategies and RNA interference (RNAi) based on transfection of single stranded DNA (ssDNA) oligomers and small interfering RNAs (siRNAs), respectively. We are targeting βgalactosidase (βgal) in K18-lacZ transgenic mice as a surrogate protein. These animals express βgal under the control of the cytokeratin 18 (K18) promoter, which has a very similar expression profile to the CF gene in airway epithelial cells. First, ssDNA and siRNAs molecules against the αENaC subunit were assessed in murine kidney cells (M1), which express high levels of ENaC and are suitable for electrophysiological analysis of ENaC activity. Ss DNA and siRNAs against βgal were assessed in NIH-3T3-lacZ cells, which stably express βgal. We designed 5 ENaCand 5 lacZssDNA (20 mers), using computer sided design. Two out of 5 ENaC AS molecules reduced mRNA expression by approximately 60% (n=6, p<0.05) and 2 out of five lacZAS reduced mRNA expression by about 70% (n=6, p<0.05) in a dose-dependent fashion. We assessed 3 ENaC siRNA molecules (none of which has been effective in reducing ENaC mRNA levels) and 10 lacZ siRNAs, of which three were effective in reducing βgal expression by at least 50% (p<0.05). The rational design of siRNA for RNAi is not well understood and emperical changes may necessary to identify the most effective molecules. We also transfected M1 cells with FITClabelled ssDNA and siRNAs and analysed intracellular distribution at different time-points after transfection (1,15,30,60min and 2,4,6,8 and 24 hrs). Although transfection efficiency was similar the intracellular distribution of these molecules was very different at all time-points. The ssDNA was visible as diffuse staining as early as 30 min after transfection and preferentially accumulated in the nuclei at all time-points, whereas the siRNAs was not visible in the nucleus at any time-point, but accumulated in the cytoplasm of transfected cells. This intracellular distribution of ssDNA and siRNA is consistent with the presumed site of action for antisense and RNAi, respectively. We have begun in vivo studies with lacZ siRNA. The lungs of K18-lacZ mice were transfected with lipid (GL67)-complexed lacZsiRNA via a standard “sniffing” protocol. Forty-eight hrs after transfection lacZ mRNA was reduced 30%, when compared to control animals (n=14, p<0.01). Interestingly, in vivo bio-distribution Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
studies using FITC-dsRNA showed no signal in airway epithelial cells 1 or 24 hrs after transfection. This may be due to degradation, with only small amounts of dsRNA being taken up into the cell. Further optimisation of RNAi and studies using antisense molecules in the lung are currently under way.
1010. Adenoviral and AAV Vectors for an Efficient Intracerebral Gene Transfer in the Sandhoff Mouse Model Christophe A. Bourgoin,1 Carla Emiliani,2 Antoinette Gelot,3 Brunela Tancini,2 Montanucci Pia,2 Livia Poenaru,1 Catherine Caillaud.1 1 Departement GDPM, Institut Cochin (INSERM, CNRS, Universite Paris 5), Paris, France; 2Biochemical Sciences and Molecular Biotechnologies Department, University of Perugia, Perugia, Italy; 3Unite de Neuropathologie, Hopital Trousseau, Paris, France. Sandhoff disease is an autosomal recessive neurodegenerative disorder characterized by the intralysosomal accumulation of GM2 ganglioside. It is due to mutations in the HEXB gene encoding the hexosaminidases β-chain and results in a hexosaminidases A (αβ) and B (ββ) deficiency. In order to test the feasibility of gene transfer methods in this model, a recombinant adenoviral vector encoding the β-subunit was injected intracerebrally into the murine model of Sandhoff disease. It leaded to a normal hexosaminidases total activity in the entire brain and a partial restoration of the Hex A specific activity. The coadministration of hyperosmotic concentrations of mannitol with the adenoviral vector resulted in a drastic enhancement of the vector diffusion in the injected hemisphere and significantly improved transduction efficiency. Our data underline the advantage of the use of mannitol in combination with low doses of adenoviral vectors, allowing a high and widespread expression without any adeno-related lesions. AAV vectors containing the human HEXA and HEXB cDNAs under the control of the CAG promoter were also constructed. AAV-HEXB was first injected alone into the brain of hexb -/neonates. Histological staining showed a high enzymatic activity in widely diffuse areas. Hexosaminidases activity was restored to the normal level in the whole brain, but Hex A levels remained low. The coadministration of both HEXA and HEXB vectors in the CNS permitted to reach therapeutic levels of hexosaminidases A and B, as demonstrated by the GM2 ganglioside degradation, confirming the necessity of supplying both subunits in the case of heterodimeric proteins, such as Hex A.
1011. Gastric Acid Neutralization and Protease Inhibition Improves Oral Recombinant AdenoAssociated Virus-Mediated Gene Delivery to the Intestines Guohong Shao,1 Kristin C. Backstrom,1 Qin Huang,1 Thomas J. Sferra.1 1 Center for Gene Therapy, Children’s Research Institute, Columbus, OH. Gene transfer to the gastrointestinal tract mediated by recombinant adeno-associated virus (rAAV) vectors has many potential applications including complementation of single gene disorders, genetic immunization and production of therapeutic molecules to act at local or distant sites. In vitro studies indicate that rAAV serotype 2 vectors are adversely affected by conditions within the GI tract (e.g. low intragastric pH, secreted proteases). The objective of this study was to evaluate whether acid neutralization and protease inhibition can improve rAAV gene delivery to the intestines. S389