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548. Ethanol Increases Transgene Expression in Parenchymal Liver Cells after Adenoviral Transfer in Rabbits
ADENOVIRUS VECTORS: APPLICATIONS 548. Ethanol Increases Transgene Expression in Parenchymal Liver Cells after Adenoviral Transfer in Rabbits
549. Peripheral Gene Delivery of Glial Cell Line-Derived Neurotrophic Factor Alleviated Neurological Deficits in Diabetic Rats
Jan Snoeys,1 Joke Lievens,1 Yingmei Feng,1 Peter Frederik,2 Désiré Collen,1 Eddie Wisse,2 Bart De Geest.1 1 Center for Molecular and Vascular Biology, Katholieke Universiteit Leuven, Leuven, Vlaams Brabant, Belgium; 2EM unit Pathology, Universiteit Maastricht, Maastricht, Netherlands.
Guei-Sheung Liu,1,2 Chiou-Lian Lai,1 Sheng-Long Howng,1 MingHong Tai.1,2 1 Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 2Dept. of Medical Education & Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.
Background: We have previously shown that the size of sinusoidal fenestrae is a critical determinant of hepatocyte transduction after adenoviral transfer (Lievens et al. Gene Therapy 2004; 11: 1523-1531). Previous in vitro studies have demonstrated that ethanol increases the size of fenestrae. The diameter of fenestrae in New Zealand White (NZW) rabbits is approximately 1.7-fold smaller than in C57BL/6 mice. Therefore, the hypothesis of this study was that intravenous administration of ethanol before transfer may increase transgene expression in NZW rabbits but not in C57BL/ 6 mice. Methods: Gene transfer of an E1E3E4-deleted vector containing the hepatocyte-specific 1.5 kb human α1-antitrypsin promoter upstream of the genomic human apo A-I sequence and 4 copies of the human apo E enhancer (AdA-I) was performed by marginal ear vein and tail vein injection in NZW rabbits and C57BL/6 mice, respectively. Isolation of parenchymal (PC) and non-parenchymal (NPC) liver cells was performed by collagenase perfusion and Nycodenz centrifugation. Transgene DNA levels were determined by real time PCR. The size of sinusoidal fenestrae was determined by transmission electron microscopy (TEM) on plastic embedded specimens. Results: Injection of 0.75 g/kg of ethanol 10 minutes before transfer with 4 x 1012 particles/kg of AdA-I in NZW rabbits increased human apo A-I plasma levels 22-fold (22 ± 5.8 mg/dl; p<0.001) compared to control NZW rabbits (1.0 ± 0.88 mg/dl). In contrast, administration of ethanol 10 minutes before transfer with 5 x 1010 particles of AdAI in C57BL/6 mice resulted in a marginal 1.3-fold (p<0.05) increase of human apo A-I levels compared to control mice. Consistent with the presence of an anatomical barrier for hepatocyte transduction in NZW rabbits, the ratio of the human apo A-I transgene DNA copy number in the PC (0.59 ± 0.13) versus transgene DNA in the NPC (2.8 ± 0.60) at day 3 after transfer was very low (0.21 ± 0.019). Injection of ethanol before transfer in NZW rabbits did no affect the transgene DNA level in the liver but increased the PC/NPC ratio 3.8-fold (0.78 ± 0.098; p<0.01), indicating a shift in the intrahepatic transgene DNA distribution. The ratio of the human apo A-I DNA copy number in the PC (3.5 ± 0.53) versus transgene DNA in the NPC (0.56 ± 0.11) at day 3 after transfer in C57BL/6 mice was 5.7 ± 0.74 and was not altered by ethanol administration. The diameter of fenestrae in NZW rabbits was 102 ± 2.1 nm and was significantly smaller than the size of adenoviral vectors determined using Vitrobot technology (93 ± 0.29 nm with superimposed fibers of 30 nm). In contrast to previous in vitro observations, ethanol did not significantly alter the diameter of fenestrae in vivo (96 ± 2.2 nm). Conclusion: Ethanol potently increases transgene expression in the liver after adenoviral transfer in NZW rabbits. The effect of ethanol on transgene expression may be mediated by a microcirculatory effect, which enhances the probability that a vector is transported through those fenestrae which are large enough for passage of vectors.
The neurological deficit in peripheral nerves is one of the major complications associated with diabetes. Deprivation of trophic factors may contribute to the pathogenesis of diabetic neuropathy while restored expression of neurotrophic factors could ameliorate such sensory abnormalities. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor that effectively prevents neuronal death and restores the synapse conduction during trauma of central nervous system. In the present study, we evaluated the therapeutic potential of GDNF gene delivery for neuropathy in diabetic rats. After injection of streptozotocin for 14 days, a reduction in nerve conduction velocity (NCV) with a concomitant increment in H-reflex was detected in diabetic rats by electrophysiological studies. Single, intramuscular (IM) injection of adenovirus encoding GDNF (Ad-GDNF) led to systemic GDNF expression for at least 35 days. Besides, the declined GDNF level in sciatic nerve was also restored by GDNF gene delivery. After gene delivery for 2-4 weeks, Ad-GDNF-treated rats exhibited a significant increment in NCV (P < 0.05) and a concomitant decrease in H-reflex latency (P < 0.05) comparing with animals of control groups. IM GDNF gene delivery was well tolerant to diabetic rats without affecting their biochemical parameters Therefore, peripheral GDNF gene delivery may hold potential for treatment of diabetic neuropathy.
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550. Adenoviral-Mediated Antioxidant Overexpression: Possible Protective Role Against Hyperglycemic Conditions in Human Aortic Endothelial Cells Aoife Duffy, Aaron Liew, Afshin Samali, Timothy O’Brien. 1 Medicine and Regenerative Medicine Institute, National University Of Ireland, Galway, Galway, Ireland. Intoduction: Increased oxidative stress is a well documented characteristic of diabetes mellitus, which plays a key role in the pathogenesis of diabetic vascular dysfunction. An increase in oxygen free radicals (O2 • -) is believed to be associated with diabetes mellitus. Efficient free radical scavenging is imperative for normal endothelial cell function and survival in diabetes mellitus. As superoxide dismutase (SOD) is responsible for scavenging O2 • - in eukaryotic cells, the aim of this study was to assess if transducing Human Aortic Enothelial Cells (HAEC) with recombinant adenovirus encoding MnSOD, CuZnSOD and Catalase, could reverse high glucose induced decrease in cell viability. Methods: HAECs exposed to adenoviral vectors encoding MnSOD (AdMnSOD), CuZnSOD (AdCuZnSOD), Catalase (AdCat) or Green Fluorescent Protein (AdGFP) or diluent (control) were cultured in normal glucose (NG)(5.5 mmol/L) or high glucose (HG) (30 mmol/L) medium. Cell viability was assessed by Methlythiazolydiphenyl-tetrazolium bromide (MTT) Assay. Transgene expression was detected by confocal microscopy in AdGFP transduced cells and by Western blot analysis for AdMnSOD/ AdCuZnSOD/ AdCat -transduced cells 48 hours post transduction. Results: From initial non-transduced cell viability assays, HAECs in HG displayed decrease in viability of 14% (n=11, P ≤ 0.01) compared to NG. Under HG conditions, exposure to CuZnSOD and MnSOD resulted in increased cell viability (versus non-transduced controls). Under NG conditions, exposure to MnSOD and CuZnSOD resulted in decreased cell viability (versus non-transduced controls). Catalase reduced viability Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
549. Peripheral Gene Delivery of Glial Cell Line-Derived Neurotrophic Factor Alleviated Neurological Deficits in Diabetic Rats
Jan Snoeys,1 Joke Lievens,1 Yingmei Feng,1 Peter Frederik,2 Désiré Collen,1 Eddie Wisse,2 Bart De Geest.1 1 Center for Molecular and Vascular Biology, Katholieke Universiteit Leuven, Leuven, Vlaams Brabant, Belgium; 2EM unit Pathology, Universiteit Maastricht, Maastricht, Netherlands.
Guei-Sheung Liu,1,2 Chiou-Lian Lai,1 Sheng-Long Howng,1 MingHong Tai.1,2 1 Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 2Dept. of Medical Education & Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.
Background: We have previously shown that the size of sinusoidal fenestrae is a critical determinant of hepatocyte transduction after adenoviral transfer (Lievens et al. Gene Therapy 2004; 11: 1523-1531). Previous in vitro studies have demonstrated that ethanol increases the size of fenestrae. The diameter of fenestrae in New Zealand White (NZW) rabbits is approximately 1.7-fold smaller than in C57BL/6 mice. Therefore, the hypothesis of this study was that intravenous administration of ethanol before transfer may increase transgene expression in NZW rabbits but not in C57BL/ 6 mice. Methods: Gene transfer of an E1E3E4-deleted vector containing the hepatocyte-specific 1.5 kb human α1-antitrypsin promoter upstream of the genomic human apo A-I sequence and 4 copies of the human apo E enhancer (AdA-I) was performed by marginal ear vein and tail vein injection in NZW rabbits and C57BL/6 mice, respectively. Isolation of parenchymal (PC) and non-parenchymal (NPC) liver cells was performed by collagenase perfusion and Nycodenz centrifugation. Transgene DNA levels were determined by real time PCR. The size of sinusoidal fenestrae was determined by transmission electron microscopy (TEM) on plastic embedded specimens. Results: Injection of 0.75 g/kg of ethanol 10 minutes before transfer with 4 x 1012 particles/kg of AdA-I in NZW rabbits increased human apo A-I plasma levels 22-fold (22 ± 5.8 mg/dl; p<0.001) compared to control NZW rabbits (1.0 ± 0.88 mg/dl). In contrast, administration of ethanol 10 minutes before transfer with 5 x 1010 particles of AdAI in C57BL/6 mice resulted in a marginal 1.3-fold (p<0.05) increase of human apo A-I levels compared to control mice. Consistent with the presence of an anatomical barrier for hepatocyte transduction in NZW rabbits, the ratio of the human apo A-I transgene DNA copy number in the PC (0.59 ± 0.13) versus transgene DNA in the NPC (2.8 ± 0.60) at day 3 after transfer was very low (0.21 ± 0.019). Injection of ethanol before transfer in NZW rabbits did no affect the transgene DNA level in the liver but increased the PC/NPC ratio 3.8-fold (0.78 ± 0.098; p<0.01), indicating a shift in the intrahepatic transgene DNA distribution. The ratio of the human apo A-I DNA copy number in the PC (3.5 ± 0.53) versus transgene DNA in the NPC (0.56 ± 0.11) at day 3 after transfer in C57BL/6 mice was 5.7 ± 0.74 and was not altered by ethanol administration. The diameter of fenestrae in NZW rabbits was 102 ± 2.1 nm and was significantly smaller than the size of adenoviral vectors determined using Vitrobot technology (93 ± 0.29 nm with superimposed fibers of 30 nm). In contrast to previous in vitro observations, ethanol did not significantly alter the diameter of fenestrae in vivo (96 ± 2.2 nm). Conclusion: Ethanol potently increases transgene expression in the liver after adenoviral transfer in NZW rabbits. The effect of ethanol on transgene expression may be mediated by a microcirculatory effect, which enhances the probability that a vector is transported through those fenestrae which are large enough for passage of vectors.
The neurological deficit in peripheral nerves is one of the major complications associated with diabetes. Deprivation of trophic factors may contribute to the pathogenesis of diabetic neuropathy while restored expression of neurotrophic factors could ameliorate such sensory abnormalities. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor that effectively prevents neuronal death and restores the synapse conduction during trauma of central nervous system. In the present study, we evaluated the therapeutic potential of GDNF gene delivery for neuropathy in diabetic rats. After injection of streptozotocin for 14 days, a reduction in nerve conduction velocity (NCV) with a concomitant increment in H-reflex was detected in diabetic rats by electrophysiological studies. Single, intramuscular (IM) injection of adenovirus encoding GDNF (Ad-GDNF) led to systemic GDNF expression for at least 35 days. Besides, the declined GDNF level in sciatic nerve was also restored by GDNF gene delivery. After gene delivery for 2-4 weeks, Ad-GDNF-treated rats exhibited a significant increment in NCV (P < 0.05) and a concomitant decrease in H-reflex latency (P < 0.05) comparing with animals of control groups. IM GDNF gene delivery was well tolerant to diabetic rats without affecting their biochemical parameters Therefore, peripheral GDNF gene delivery may hold potential for treatment of diabetic neuropathy.
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550. Adenoviral-Mediated Antioxidant Overexpression: Possible Protective Role Against Hyperglycemic Conditions in Human Aortic Endothelial Cells Aoife Duffy, Aaron Liew, Afshin Samali, Timothy O’Brien. 1 Medicine and Regenerative Medicine Institute, National University Of Ireland, Galway, Galway, Ireland. Intoduction: Increased oxidative stress is a well documented characteristic of diabetes mellitus, which plays a key role in the pathogenesis of diabetic vascular dysfunction. An increase in oxygen free radicals (O2 • -) is believed to be associated with diabetes mellitus. Efficient free radical scavenging is imperative for normal endothelial cell function and survival in diabetes mellitus. As superoxide dismutase (SOD) is responsible for scavenging O2 • - in eukaryotic cells, the aim of this study was to assess if transducing Human Aortic Enothelial Cells (HAEC) with recombinant adenovirus encoding MnSOD, CuZnSOD and Catalase, could reverse high glucose induced decrease in cell viability. Methods: HAECs exposed to adenoviral vectors encoding MnSOD (AdMnSOD), CuZnSOD (AdCuZnSOD), Catalase (AdCat) or Green Fluorescent Protein (AdGFP) or diluent (control) were cultured in normal glucose (NG)(5.5 mmol/L) or high glucose (HG) (30 mmol/L) medium. Cell viability was assessed by Methlythiazolydiphenyl-tetrazolium bromide (MTT) Assay. Transgene expression was detected by confocal microscopy in AdGFP transduced cells and by Western blot analysis for AdMnSOD/ AdCuZnSOD/ AdCat -transduced cells 48 hours post transduction. Results: From initial non-transduced cell viability assays, HAECs in HG displayed decrease in viability of 14% (n=11, P ≤ 0.01) compared to NG. Under HG conditions, exposure to CuZnSOD and MnSOD resulted in increased cell viability (versus non-transduced controls). Under NG conditions, exposure to MnSOD and CuZnSOD resulted in decreased cell viability (versus non-transduced controls). Catalase reduced viability Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy