- Email: [email protected]
142. Serotype Optimization of an AAV Vector for Gene Therapy in Canine and Murine Glycogen Storage Disease Type Ia (GSD-Ia)
GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY I 140. Gene Therapy Approaches Combining the Immunomodulatory Properties of Liver and IGF-I To Prevent Type I Diabetes
Sabrina Tafuro,1,2 Xavier M. Anguela,1,2 Judith Agudo,1,2 Alba Casellas,1,2 David Callejas,1,2 Mercè Obach,1,2 Carles Roca,1,2 Albert Ruzo,1,2 Fàtima Bosch.1,2 1 Center of Animal Biotechnology and Gene Therapy (CBATEG) and Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain; 2CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain. In type 1 diabetes (T1D), the loss of tolerance to pancreatic ß cell antigens results in a T cell-dependent autoimmune destruction of insulin-producing ß-cells, leading to insulin deciency. Thus, restoration of tolerance to self-antigens and control of autoreactive T lymphocytes is required to prevent T1D. The liver has unique immunomodulatory properties and hepatic gene transfer can induce tolerance to therapeutic proteins and suppress autoimmune diseases, in part by regulatory T cell (Treg) activation. Insulin-like growth factor-I (IGF-I) prevents T1D when expressed in ß-cells of transgenic mice or subcutaneously injected. Therefore, the aim of this study was to combine the immunomodulatory properties of the liver with the protective effects of IGF-I to develop a non-viral, liver-directed gene therapy approach for type 1 diabetes. Viral gene therapy approaches may elicit sustained transgene expression that would not be shut down upon achievement of desirable tolerance. In contrast, hydrodynamic tail vein (HTV) injection of naked plasmid DNA renders transient the expression of a therapeutic gene in liver cells. By using HTV injection of a GFP-expressing plasmid, we demonstrated that in addition to hepatocytes, liver non-parenchymal cells (NPCs) could be transfected and were able to express foreign genes. With the same technique, we tested whether efcient expression of IGF-I either in hepatocytes or in liver non-parenchymal cells could lead to prevention of T1D. HTV injection of a plasmid expressing IGF-I under the control of either an ubiquitous or a myeloid-specic promoter decreased the incidence of diabetes in a mouse model of the disease expressing human interferon-ß in ß-cells (TgIFN-ß), reaching permanent protection after ten administrations of IGF-I plasmid. When IGF-I expression was limited to hepatocytes by using a hepatocyte-specic promoter, mice developed overt diabetes. Our results suggested that IGF-1 transiently overexpressed in mouse liver non-parenchymal cells might be a key process in mediating protection from T1D. We also showed that Treg cells may play a central role in disease prevention suggesting IGF-I-mediated induction of Tregs as a key mechanism to protect mice from diabetes. Overall, this study indicates that non-viral, liver-targeted expression of IGF-I may represent a new approach to prevent T1D. Thus, gene therapy approaches combining the liver immunomodulatory properties with IGF-I protective effects may prove successful to control T1D and other autoimmune diseases.
cell culture conditions have not allowed extensive proliferation of hepatocytes, establishment of hepatocyte proliferation system that can be used for gene transduction has been be highly desired. The present study was designed to determine whether hemophilia hepatocytes could be efciently propagated in the liver of uPA/SCID mice. Once the full reconstitution of the uPA/SCID liver with hemophilia hepatocytes is successfully achieved, AAV-mediated gene therapy was conducted to the mice to determine whether the uPA/SCID mouse propagation system would offer living system to transduce coagulation factor gene to the hemophilia hepatocytes. [Experimental methods] Hemophilia hepatocytes were isolated from factor IX knockout (FIX-KO) mice. Isolated cells were then puried by Percoll isodensity purication. A total of 5 x 105 viable hepatocytes were transplanted into the liver of uPA/SCID mice through splenic injection and the liver repopulation status with FIXKO hepatocytes were assessed. After the complete repopulation of the uPA/SCID liver with the FIX-KO hepatocytes was conrmed, AAV vector (AAV8-hF.IX16) was injected through the tail vein at a dose of 7.5 x 1012 vg/mouse. Blood samples were periodically collected for the assessment of plasma hFIX activity as well as hFIX antigen levels. [Results] The plasma FIX activity of the recipient uPA/SCID mice showed progressive decreases after FIX-KO hepatocyte transplantation. At week 8 or later, the FIX activity levels became undetectable (> 0.5% of normal mouse plasma). We then isolated hepatocytes at week 12 from the recipient livers for the genomic analyses and found that FIX-KO hepatocytes constituted more than 99.5 % of the recipient uPA/SCID livers. These results conrmed that the transplanted FIX-KO hepatocytes actively proliferated to fully reconstitute the uPA/SCID livers. After the AAV infusion, the FIX-KO hepatocyte-repopulated uPA/SCID mice showed persistent high levels of plasma hFIX levels (> 50 ug/ml) throughout 8-week experimental period at similar hFIX levels observed in the AAVinjected naïve FIX-KO mice. [Conclusions] The present studies demonstrated that fully reconstituted hemophilic mouse livers can be generated by transplantation of hemophilia hepatocytes into the liver of uPA/ SCID mice. It is also of note that the reconstituted hemophilic mouse livers were efciently transduced with AAV infusion. Since we have previously documented that human hepatocytes are able to reconstitute the uPA/SCID livers and hepatocytes are able to be recovered, the living mouse system can be a potential viable method to generate gene-corrected autologous hepatocyte and liver tissue source for hepatocyte-based therapies toward inherited liver diseases including hemophilia.
142. Serotype Optimization of an AAV Vector for Gene Therapy in Canine and Murine Glycogen Storage Disease Type Ia (GSD-Ia)
141. Hepatocyte Engineering and Genetic Modication Approaches of Hemophilia Hepatocytes within a Living Mouse
Xiaoyan Luo,1 Amanda K. Demaster,1,2 Songtao Li,1 Kyha Williams,2 Talmage T. Brown,3 Daniel M. Kozink,1 Andrew Bird,1 Dwight D. Koeberl.1 1 Pediatrics, Duke University Medical Center, Durham, NC; 2 Laboratory Animal Resources, Duke University Medical Center, Durham, NC; 3North Carolina State University, College of Veterinary Medicine, Durham, NC.
[Background] Hepatocyte has been considered as a valuable target cell type for cell and gene therapy toward hemophilia. Since present
Glycogen storage disease type Ia (GSD-Ia) described in dogs closely resembles human GSD-Ia. GSD-Ia human and canine patients suffer from complications associated with glucose-6-phosphatase (G6Pase) deciency (hypoglycemia, hyperlipidemia, growth retardation, and early death). For the past two decades survival of human patients that are placed under intensive nutritional management with uncooked corn starch has improved; however, long-term complications persist including renal failure, nephrolithiasis, hepatic adenomas, and a high risk for hepatocellular carcinoma. We developed a 2.3 kbp genome
Kazuo Ohashi,1 Kohei Tatsumi,1 Chise Tateno,2 Hiroyuki Nakai,3 Anja Ehrhardt,4 Rie Utoh,1 Katsutoshi Yoshizato,2 Teruo Okano.1 1 Inst. of Adv Biomed Eng and Sci, Tokyo Women’s Medical University, Shinjyuku, Tokyo, Japan; 2PhoenixBio, Higashihiroshima, Japan; 3Dept Microbiol and Mol Genet, Univ of Pittsburgh School of Medicine, Pittsburgh, PA; 4Max von Pettenkofer-Insitute, Univ of Munich, Munich, Germany.
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Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY I AAV vector containing a minimal human G6Pase promoter to drive regulated G6Pase expression in vivo. A single administration of the AAV2/8 vector to young GSD-Ia mice and dogs accomplished the correction of blood glucose and cholesterol for >one year. The correction of hypoglycemia during prolonged fasting, for >6 hours, provided further evidence of efcacy in GSD-Ia dogs, and this correction has been sustained at 2.5 years of age in two dogs. Thus, the AAV2/8 vector encoding human G6Pase prevented hypoglycemia during fasting, the primary metabolic abnormality in GSD-Ia; however the number of vector particles needed was higher than what has been administered in clinical trials (1x1013 vp/kg). Previously 2x1012 vp/kg of an AAV2/2 vector was administered in the hemophilia B trial before anti-capsid immune responses were encountered. We have evaluated AAV2/2, AAV2/7, and AAV2/9 pseudotypes of the AAV vector in G6Pase (-/-) mice after administration of 2x1012 vp/ kg. Normal blood glucose for 8 hours duration fasting in absence of any cornstarch therapy would represent an effective cure of GSD-Ia, because intermittent cornstarch therapy would not be needed. For AAV2/9 vector-treated G6Pase (-/-) mice that received 1x1013 vp/kg blood glucose was equivalent to unaffected littermates following the 8 hour fast at 6 months of age, and blood glucose was only slightly reduced but in the normal range at 12 months of age following 1x1013 vp/kg. The more remarkable demonstration of efcacy was that G6Pase (-/-) mice treated with only 2x1012 vp/kg of the AAV2/9 vector, blood glucose was normal after 8 hours fasting at 6 months of age. Furthermore, the AAV2/9 vector transduced both liver and kidney with higher efciency than other pseudotypes, as demonstrated by the correction of G6Pase deciency and the reduction of glycogen accumulations in both of these tissues. Recently, the evaluation of the rst GSD-Ia puppy treated with this AAV2/9 vector revealed normal blood glucose throughout >8 hours of fasting at 5 months of age. In summary, this AAV2/9 vector encoding human G6Pase has emerged as a leading candidate for gene therapy in GSD-Ia.
143. Systemic Delivery of Bioactive GlucagonLike Peptide 1 Following Adenoviral VectorMediated Gene Transfer to Murine Submandibular Glands
Antonis Voutetakis,1 Anne M. Rowzee,1 Ana P. Cotrim,1 Changyu Zheng,1 Trushar Rathod,2 Tulin Yanik,2 Y. P. Loh,2 Bruce J. Baum,1 Niamh X. Cawley.2 1 Molecular Physiology and Therapeutics Branch, NIDCR, NIH, Bethesda, MD; 2Section on Cellular Neurobiology, Eunice Kennedy Shriver NICHD, NIH, Bethesda, MD. Glucagon-like peptide 1 (GLP-1) is a 37 amino acid peptide that is expressed as part of the prohormone, proglucagon. In the gut endocrine cells, proglucagon is cleaved to generate GLP-1, which is further processed to generate an amidated C-terminus. The N-terminus is also cleaved to generate GLP-1(7-36-amide), which is the bioactive form of GLP-1 released into circulation after a meal. GLP-1 acts as an incretin and functions to slow gastric emptying and to enhance insulin function by increasing insulin secretion from pancreatic β-cells and increasing β-cell mass. Its biological half-life is short, approximately 2-3 min, and it is inactivated by dipeptidylaminopeptidase IV (DPP IV) in the blood. Because of its potential use in the treatment of Type 2 diabetes many approaches are being used to enhance its effectiveness, including generating non-cleavable GLP-1 analogues, DPP IV inhibitors, longer lasting GLP-1 receptor agonists and sustainable and ultimately regulatable levels of GLP-1 by gene therapy. A serotype 5 adenoviral construct encoding GLP1 (Ad-GLP-1) was generated and initially shown to direct GLP-1 production in vitro. The expression cassette was engineered with the signal sequence of mouse growth hormone, to direct GLP-1 into the secretory pathway, followed by a furin cleavage site and the GLP1(7-36) sequence. In vitro analysis of the expressed transgenic GLP-1 Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
demonstrated that it was more resistant to degradation by DPP IV, due to an Ala to Gly substitution at position 8, and it was biologically active, since it could specically induce the release of insulin from the NIT1 pancreatic β-cell line. In vivo studies demonstrated that mice administered the Ad-GLP-1 vector in their submandibular glands had serum levels of GLP-1 approximately 3-times higher than control mice transduced with an Ad-Luciferase (Ad-Luc) vector. In healthy, fasted animals serum glucose levels were similar between Ad-GLP-1 and Ad-Luc treated mice, in keeping with GLP-1’s glucose dependent action. However, when challenged with glucose in a glucose tolerance test, Ad-GLP-1 treated healthy mice lowered serum levels of glucose signicantly faster than the Ad-Luc-treated mice. Finally, in a mouse model of induced diabetes mellitus using alloxan, progression of hyperglycemia was signicantly attenuated in the mice pre-treated (-24 h) with the Ad-GLP-1 vector to their submandibular glands compared to control mice treated with the Ad-Luc vector. These studies demonstrate that bioactive GLP1, normally secreted from endocrine cells through the regulated secretory pathway, can be secreted into the bloodstream following gene transfer to exocrine salivary gland cells. The transgenic GLP-1 affects glucose homeostasis and suggests a potential application for the treatment of type 2 diabetes.
144. A Non-Viral, GNE-Lipoplex Treatment to Correct Sialylation Defects in Gne-Mutant (M712T) Mice
Tal Yardeni,1,4 Carla Ciccone,1 Shelley Hoogstraten-Miller,2 Daniel Darvish,3 Yair Anikster,4 Phil Maples,5 Chris Jay,5 William A. Gahl,1 John Nemunaitis,5,6 Marjan Huizing.1 1 Medical Genetics Branch, NHGRI, NIH, Bethesda, MD; 2Ofce of Laboratory Animal Medicine, NHGRI, NIH, Bethesda, MD; 3 HIBM Research Group, Encino, Encino, CA; 4Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 5Gradalis Inc., Gradalis Inc., Dallas, TX; 6Mary Crowley Cancer Research Centers, Dallas, TX. Hereditary Inclusion Body Myopathy (HIBM) is an adult-onset, progressive neuromuscular disorder caused by GNE mutations. GNE encodes the ubiquitously expressed, key enzyme in sialic acid (SA) synthesis, UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE/MNK). Decreased SA production consequently leads to reduced sialylation of a variety of glycoproteins. We created a Gne knock-in (M712T) mouse to establish an HIBM animal model. Surprisingly, mutant mice died before day 3 of life (P3) from severe glomerulopathy. Renal ndings included segmental splitting of the glomerular basement membrane, effacement of podocyte foot processes, and reduced sialylation of the major podocyte sialoprotein, podocalyxin. Oral administration of the SA precursor, ManNAc, yielded survival beyond P3 in 43% of the mutant pups. Survivors exhibited increased sialylation of podocalyxin, increased Gne/Mnk protein and increased Gne enzyme activities. To investigate GNE gene therapy as an alternative to oral ManNAc therapy, mice were injected with human hGNE-Lipoplex at P1 via intra-hepatic or retro-orbital (IV) injection. The routes of delivery were proven to be safe and well-tolerated. Mice receiving intra-hepatic hGNE-Lipoplex had signicant levels (500 – 50,000 fg) of hGNE plasmid DNA and hGNE RNA expression in the liver, with a small level (∼50 fg) of plasmid DNA detected in the skeletal muscle. No hGNE plasmid DNA was detected in the kidneys of pups receiving intra-hepatic delivery. Mice receiving retro-orbital delivery had signicant levels (500 – 10,000 fg) of hGNE plasmid DNA, and hGNE RNA expression was detected in liver, kidney, and skeletal muscle. One mutant pup surviving beyond P3 exhibited increased sialylation of podocalyxin at P5. Based on the results from this pilot study and the fact that the renal phenotype begins during the embryonic stage, a pregnant female mouse was injected retro-orbitally at embryonic day 13 (E13) and the embryos were collected at E19. Western blots S55
Sabrina Tafuro,1,2 Xavier M. Anguela,1,2 Judith Agudo,1,2 Alba Casellas,1,2 David Callejas,1,2 Mercè Obach,1,2 Carles Roca,1,2 Albert Ruzo,1,2 Fàtima Bosch.1,2 1 Center of Animal Biotechnology and Gene Therapy (CBATEG) and Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain; 2CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain. In type 1 diabetes (T1D), the loss of tolerance to pancreatic ß cell antigens results in a T cell-dependent autoimmune destruction of insulin-producing ß-cells, leading to insulin deciency. Thus, restoration of tolerance to self-antigens and control of autoreactive T lymphocytes is required to prevent T1D. The liver has unique immunomodulatory properties and hepatic gene transfer can induce tolerance to therapeutic proteins and suppress autoimmune diseases, in part by regulatory T cell (Treg) activation. Insulin-like growth factor-I (IGF-I) prevents T1D when expressed in ß-cells of transgenic mice or subcutaneously injected. Therefore, the aim of this study was to combine the immunomodulatory properties of the liver with the protective effects of IGF-I to develop a non-viral, liver-directed gene therapy approach for type 1 diabetes. Viral gene therapy approaches may elicit sustained transgene expression that would not be shut down upon achievement of desirable tolerance. In contrast, hydrodynamic tail vein (HTV) injection of naked plasmid DNA renders transient the expression of a therapeutic gene in liver cells. By using HTV injection of a GFP-expressing plasmid, we demonstrated that in addition to hepatocytes, liver non-parenchymal cells (NPCs) could be transfected and were able to express foreign genes. With the same technique, we tested whether efcient expression of IGF-I either in hepatocytes or in liver non-parenchymal cells could lead to prevention of T1D. HTV injection of a plasmid expressing IGF-I under the control of either an ubiquitous or a myeloid-specic promoter decreased the incidence of diabetes in a mouse model of the disease expressing human interferon-ß in ß-cells (TgIFN-ß), reaching permanent protection after ten administrations of IGF-I plasmid. When IGF-I expression was limited to hepatocytes by using a hepatocyte-specic promoter, mice developed overt diabetes. Our results suggested that IGF-1 transiently overexpressed in mouse liver non-parenchymal cells might be a key process in mediating protection from T1D. We also showed that Treg cells may play a central role in disease prevention suggesting IGF-I-mediated induction of Tregs as a key mechanism to protect mice from diabetes. Overall, this study indicates that non-viral, liver-targeted expression of IGF-I may represent a new approach to prevent T1D. Thus, gene therapy approaches combining the liver immunomodulatory properties with IGF-I protective effects may prove successful to control T1D and other autoimmune diseases.
cell culture conditions have not allowed extensive proliferation of hepatocytes, establishment of hepatocyte proliferation system that can be used for gene transduction has been be highly desired. The present study was designed to determine whether hemophilia hepatocytes could be efciently propagated in the liver of uPA/SCID mice. Once the full reconstitution of the uPA/SCID liver with hemophilia hepatocytes is successfully achieved, AAV-mediated gene therapy was conducted to the mice to determine whether the uPA/SCID mouse propagation system would offer living system to transduce coagulation factor gene to the hemophilia hepatocytes. [Experimental methods] Hemophilia hepatocytes were isolated from factor IX knockout (FIX-KO) mice. Isolated cells were then puried by Percoll isodensity purication. A total of 5 x 105 viable hepatocytes were transplanted into the liver of uPA/SCID mice through splenic injection and the liver repopulation status with FIXKO hepatocytes were assessed. After the complete repopulation of the uPA/SCID liver with the FIX-KO hepatocytes was conrmed, AAV vector (AAV8-hF.IX16) was injected through the tail vein at a dose of 7.5 x 1012 vg/mouse. Blood samples were periodically collected for the assessment of plasma hFIX activity as well as hFIX antigen levels. [Results] The plasma FIX activity of the recipient uPA/SCID mice showed progressive decreases after FIX-KO hepatocyte transplantation. At week 8 or later, the FIX activity levels became undetectable (> 0.5% of normal mouse plasma). We then isolated hepatocytes at week 12 from the recipient livers for the genomic analyses and found that FIX-KO hepatocytes constituted more than 99.5 % of the recipient uPA/SCID livers. These results conrmed that the transplanted FIX-KO hepatocytes actively proliferated to fully reconstitute the uPA/SCID livers. After the AAV infusion, the FIX-KO hepatocyte-repopulated uPA/SCID mice showed persistent high levels of plasma hFIX levels (> 50 ug/ml) throughout 8-week experimental period at similar hFIX levels observed in the AAVinjected naïve FIX-KO mice. [Conclusions] The present studies demonstrated that fully reconstituted hemophilic mouse livers can be generated by transplantation of hemophilia hepatocytes into the liver of uPA/ SCID mice. It is also of note that the reconstituted hemophilic mouse livers were efciently transduced with AAV infusion. Since we have previously documented that human hepatocytes are able to reconstitute the uPA/SCID livers and hepatocytes are able to be recovered, the living mouse system can be a potential viable method to generate gene-corrected autologous hepatocyte and liver tissue source for hepatocyte-based therapies toward inherited liver diseases including hemophilia.
142. Serotype Optimization of an AAV Vector for Gene Therapy in Canine and Murine Glycogen Storage Disease Type Ia (GSD-Ia)
141. Hepatocyte Engineering and Genetic Modication Approaches of Hemophilia Hepatocytes within a Living Mouse
Xiaoyan Luo,1 Amanda K. Demaster,1,2 Songtao Li,1 Kyha Williams,2 Talmage T. Brown,3 Daniel M. Kozink,1 Andrew Bird,1 Dwight D. Koeberl.1 1 Pediatrics, Duke University Medical Center, Durham, NC; 2 Laboratory Animal Resources, Duke University Medical Center, Durham, NC; 3North Carolina State University, College of Veterinary Medicine, Durham, NC.
[Background] Hepatocyte has been considered as a valuable target cell type for cell and gene therapy toward hemophilia. Since present
Glycogen storage disease type Ia (GSD-Ia) described in dogs closely resembles human GSD-Ia. GSD-Ia human and canine patients suffer from complications associated with glucose-6-phosphatase (G6Pase) deciency (hypoglycemia, hyperlipidemia, growth retardation, and early death). For the past two decades survival of human patients that are placed under intensive nutritional management with uncooked corn starch has improved; however, long-term complications persist including renal failure, nephrolithiasis, hepatic adenomas, and a high risk for hepatocellular carcinoma. We developed a 2.3 kbp genome
Kazuo Ohashi,1 Kohei Tatsumi,1 Chise Tateno,2 Hiroyuki Nakai,3 Anja Ehrhardt,4 Rie Utoh,1 Katsutoshi Yoshizato,2 Teruo Okano.1 1 Inst. of Adv Biomed Eng and Sci, Tokyo Women’s Medical University, Shinjyuku, Tokyo, Japan; 2PhoenixBio, Higashihiroshima, Japan; 3Dept Microbiol and Mol Genet, Univ of Pittsburgh School of Medicine, Pittsburgh, PA; 4Max von Pettenkofer-Insitute, Univ of Munich, Munich, Germany.
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Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY I AAV vector containing a minimal human G6Pase promoter to drive regulated G6Pase expression in vivo. A single administration of the AAV2/8 vector to young GSD-Ia mice and dogs accomplished the correction of blood glucose and cholesterol for >one year. The correction of hypoglycemia during prolonged fasting, for >6 hours, provided further evidence of efcacy in GSD-Ia dogs, and this correction has been sustained at 2.5 years of age in two dogs. Thus, the AAV2/8 vector encoding human G6Pase prevented hypoglycemia during fasting, the primary metabolic abnormality in GSD-Ia; however the number of vector particles needed was higher than what has been administered in clinical trials (1x1013 vp/kg). Previously 2x1012 vp/kg of an AAV2/2 vector was administered in the hemophilia B trial before anti-capsid immune responses were encountered. We have evaluated AAV2/2, AAV2/7, and AAV2/9 pseudotypes of the AAV vector in G6Pase (-/-) mice after administration of 2x1012 vp/ kg. Normal blood glucose for 8 hours duration fasting in absence of any cornstarch therapy would represent an effective cure of GSD-Ia, because intermittent cornstarch therapy would not be needed. For AAV2/9 vector-treated G6Pase (-/-) mice that received 1x1013 vp/kg blood glucose was equivalent to unaffected littermates following the 8 hour fast at 6 months of age, and blood glucose was only slightly reduced but in the normal range at 12 months of age following 1x1013 vp/kg. The more remarkable demonstration of efcacy was that G6Pase (-/-) mice treated with only 2x1012 vp/kg of the AAV2/9 vector, blood glucose was normal after 8 hours fasting at 6 months of age. Furthermore, the AAV2/9 vector transduced both liver and kidney with higher efciency than other pseudotypes, as demonstrated by the correction of G6Pase deciency and the reduction of glycogen accumulations in both of these tissues. Recently, the evaluation of the rst GSD-Ia puppy treated with this AAV2/9 vector revealed normal blood glucose throughout >8 hours of fasting at 5 months of age. In summary, this AAV2/9 vector encoding human G6Pase has emerged as a leading candidate for gene therapy in GSD-Ia.
143. Systemic Delivery of Bioactive GlucagonLike Peptide 1 Following Adenoviral VectorMediated Gene Transfer to Murine Submandibular Glands
Antonis Voutetakis,1 Anne M. Rowzee,1 Ana P. Cotrim,1 Changyu Zheng,1 Trushar Rathod,2 Tulin Yanik,2 Y. P. Loh,2 Bruce J. Baum,1 Niamh X. Cawley.2 1 Molecular Physiology and Therapeutics Branch, NIDCR, NIH, Bethesda, MD; 2Section on Cellular Neurobiology, Eunice Kennedy Shriver NICHD, NIH, Bethesda, MD. Glucagon-like peptide 1 (GLP-1) is a 37 amino acid peptide that is expressed as part of the prohormone, proglucagon. In the gut endocrine cells, proglucagon is cleaved to generate GLP-1, which is further processed to generate an amidated C-terminus. The N-terminus is also cleaved to generate GLP-1(7-36-amide), which is the bioactive form of GLP-1 released into circulation after a meal. GLP-1 acts as an incretin and functions to slow gastric emptying and to enhance insulin function by increasing insulin secretion from pancreatic β-cells and increasing β-cell mass. Its biological half-life is short, approximately 2-3 min, and it is inactivated by dipeptidylaminopeptidase IV (DPP IV) in the blood. Because of its potential use in the treatment of Type 2 diabetes many approaches are being used to enhance its effectiveness, including generating non-cleavable GLP-1 analogues, DPP IV inhibitors, longer lasting GLP-1 receptor agonists and sustainable and ultimately regulatable levels of GLP-1 by gene therapy. A serotype 5 adenoviral construct encoding GLP1 (Ad-GLP-1) was generated and initially shown to direct GLP-1 production in vitro. The expression cassette was engineered with the signal sequence of mouse growth hormone, to direct GLP-1 into the secretory pathway, followed by a furin cleavage site and the GLP1(7-36) sequence. In vitro analysis of the expressed transgenic GLP-1 Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
demonstrated that it was more resistant to degradation by DPP IV, due to an Ala to Gly substitution at position 8, and it was biologically active, since it could specically induce the release of insulin from the NIT1 pancreatic β-cell line. In vivo studies demonstrated that mice administered the Ad-GLP-1 vector in their submandibular glands had serum levels of GLP-1 approximately 3-times higher than control mice transduced with an Ad-Luciferase (Ad-Luc) vector. In healthy, fasted animals serum glucose levels were similar between Ad-GLP-1 and Ad-Luc treated mice, in keeping with GLP-1’s glucose dependent action. However, when challenged with glucose in a glucose tolerance test, Ad-GLP-1 treated healthy mice lowered serum levels of glucose signicantly faster than the Ad-Luc-treated mice. Finally, in a mouse model of induced diabetes mellitus using alloxan, progression of hyperglycemia was signicantly attenuated in the mice pre-treated (-24 h) with the Ad-GLP-1 vector to their submandibular glands compared to control mice treated with the Ad-Luc vector. These studies demonstrate that bioactive GLP1, normally secreted from endocrine cells through the regulated secretory pathway, can be secreted into the bloodstream following gene transfer to exocrine salivary gland cells. The transgenic GLP-1 affects glucose homeostasis and suggests a potential application for the treatment of type 2 diabetes.
144. A Non-Viral, GNE-Lipoplex Treatment to Correct Sialylation Defects in Gne-Mutant (M712T) Mice
Tal Yardeni,1,4 Carla Ciccone,1 Shelley Hoogstraten-Miller,2 Daniel Darvish,3 Yair Anikster,4 Phil Maples,5 Chris Jay,5 William A. Gahl,1 John Nemunaitis,5,6 Marjan Huizing.1 1 Medical Genetics Branch, NHGRI, NIH, Bethesda, MD; 2Ofce of Laboratory Animal Medicine, NHGRI, NIH, Bethesda, MD; 3 HIBM Research Group, Encino, Encino, CA; 4Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 5Gradalis Inc., Gradalis Inc., Dallas, TX; 6Mary Crowley Cancer Research Centers, Dallas, TX. Hereditary Inclusion Body Myopathy (HIBM) is an adult-onset, progressive neuromuscular disorder caused by GNE mutations. GNE encodes the ubiquitously expressed, key enzyme in sialic acid (SA) synthesis, UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE/MNK). Decreased SA production consequently leads to reduced sialylation of a variety of glycoproteins. We created a Gne knock-in (M712T) mouse to establish an HIBM animal model. Surprisingly, mutant mice died before day 3 of life (P3) from severe glomerulopathy. Renal ndings included segmental splitting of the glomerular basement membrane, effacement of podocyte foot processes, and reduced sialylation of the major podocyte sialoprotein, podocalyxin. Oral administration of the SA precursor, ManNAc, yielded survival beyond P3 in 43% of the mutant pups. Survivors exhibited increased sialylation of podocalyxin, increased Gne/Mnk protein and increased Gne enzyme activities. To investigate GNE gene therapy as an alternative to oral ManNAc therapy, mice were injected with human hGNE-Lipoplex at P1 via intra-hepatic or retro-orbital (IV) injection. The routes of delivery were proven to be safe and well-tolerated. Mice receiving intra-hepatic hGNE-Lipoplex had signicant levels (500 – 50,000 fg) of hGNE plasmid DNA and hGNE RNA expression in the liver, with a small level (∼50 fg) of plasmid DNA detected in the skeletal muscle. No hGNE plasmid DNA was detected in the kidneys of pups receiving intra-hepatic delivery. Mice receiving retro-orbital delivery had signicant levels (500 – 10,000 fg) of hGNE plasmid DNA, and hGNE RNA expression was detected in liver, kidney, and skeletal muscle. One mutant pup surviving beyond P3 exhibited increased sialylation of podocalyxin at P5. Based on the results from this pilot study and the fact that the renal phenotype begins during the embryonic stage, a pregnant female mouse was injected retro-orbitally at embryonic day 13 (E13) and the embryos were collected at E19. Western blots S55