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671. Nerve Growth Factor and Neurotrophin-3 Delivered by HSV Gene Transfer Reverses Diabetic Neuropathy
GENE THERAPY FOR CONNECTIVE TISSUE compared to normal controls. Therefore, expression of Shh and of its transcriptional target Ptch is induced in murine retinal neovascularization, whether purely hypoxic (ROP) or laser-induced (CNV). To investigate whether the activation of the Shh pathway plays a role in ocular neovascularization we tested the effect of the selective Shh inhibitor cyclopamine on the hypoxia-induced angiogenesis in the ROP murine model. Systemic (subcutaneous) administration of cyclopamine resulted in down-regulation of the Shh pathway and significantly inhibited neovascularization in the ROP. To investigate the potential role of the Shh pathway during the development of the physiological retinal vasculature, cyclopamine was administered to C57/BL6 mice between P1 and P4. This resulted in significant reduction of the extent of the retinal vascular area as assessed by angiography at P5, demonstrating that the Shh pathway is required for retinal vascular development. In conclusion our results suggest that Shh plays a central role in physiological and pathological retinal angiogenesis, thus allowing designing gene based therapies to target the Shh signaling for the treatment of ocular neovascularization.
670. Mutation Independent Silencing of Genes Involved in Retinal Degeneration by RNA Interference (RNAi) and Trans Complementation by a Codon Exchanged RNAi-Resistant Transgene Siobhan Cashman, Erin Binkley, Rajendra Kumar-Singh. 1 Ophthalmology and Human Genetics, University of Utah, Salt Lake City, UT. More than one hundred different mutations in the gene encoding rhodopsin are associated with a group of retinal degenerations including retinitis pigmentosa, congenital stationary night blindness and retinitis punctata albescens. Given this large heterogeneity of mutations, it would be ideal to develop mutation independent therapies for these diseases. We describe use of the endogenous RNA interference (RNAi) machinery present in eukaryotic cells to silence mutant and wild type rhodopsin alleles by a vector expressing a short hairpin RNA (shRNA). We found that in the human embryonic retinoblast cell line 911, wild type human rhodopsin driven by a CMV promoter could be silenced by 94.34±2.17% and the most common rhodopsin-associated mutation in the United States, P23H, could be silenced by 94.9±1.9% by a shRNA targeting rhodopsin. When the degeneracy of the genetic code was used to exchange 10 of 21 nucleotides of the region that encodes for the shRNA target in human rhodopsin cDNA, we found that the shRNA was no longer able to silence the codon exchanged rhodopsin mRNA (cmRNA). Moreover, the efficiency of translation of this cmRNA was equivalent to wild type rhodopsin mRNA. For proof of principle of treatment of autosomal dominant diseases using RNAi, we cotransfected mutant (P23H) rhodopsin cDNA with a rhodopsin cDNA engineered to code for a cmRNA and a shRNA into human embryonic retinoblast 911 cells and found specific silencing of the P23H rhodopsin by 90.64±5.19%. In addition, we present data on two alternative shRNA sequences targeting human rhodopsin, one of which also silences rhodopsin albeit less efficiently than the one described above. We have cloned each of these shRNA transcripts into AAV vectors and begun testing the potential of using this strategy in animal models of retinal degeneration. In conclusion, we have identified an extremely potent shRNA sequence and a strategy to evade shRNA mediated silencing of mRNA that has the potential to be used for the treatment of a very large variety of retinal degenerations in a mutation independent manner.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
671. Nerve Growth Factor and Neurotrophin-3 Delivered by HSV Gene Transfer Reverses Diabetic Neuropathy James R. Goss,1 Munmun Chattopadhyay,2 William F. Goins,1 Darren Wolfe,1 Marina Mata,2 Joseph C. Glorioso,1 David J. Fink.2 1 Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA; 2Neurology, University of Michigan, Ann Arbor, MI. Peripheral neuropathy, involving motor, sensory, and autonomic nerves, is a common and debilitating complication of diabetes. Many features of human diabetic neuropathy can be modeled in the mouse using streptozotocin (STZ) to selectively destroy pancreatic beta cells resulting in sustained hyperglycemia but not requiring insulin replacement. We have previously demonstrated that recombinant replication defective herpes simplex virus (HSV)-based vectors expressing the neurotrophic proteins NGF (vector SHN) or NT3 (vector QL2HNT3) delivered by subcutaneous inoculation to transduce dorsal root ganglia prevents the development of neuropathy in the STZ mouse. Using a double-blind experiment, we have extended these findings by examining if these vectors could reverse STZ-induced neuropathy once it has been established. Male Swiss-Webster mice were injected with streptozotocin (STZ) and six weeks later the presence of peripheral neuropathy was confirmed by electrophysiological measurements of a foot sensory nerve response to an evoked potential and the foot withdrawal latency from a heat source. STZ-treated mice were then randomized to four treatment groups: 1) No vector; 2) vector SHZ (control vector); 3) vector SHN; or, 4) vector QL2HNT3. Mice injected with STZ developed a peripheral neuropathy characterized by a decrease in the foot nerve sensory amplitude, an increase in the foot withdrawal latency from a heat source, and a decrease in the number of nerve fibers innervating the skin of the hind foot. These indices of neuropathy were reversed 5 weeks after mice were inoculated with either SHN or QL2HNT3 into the hind foot; mice inoculated with a control vector (SHZ) demonstrated no improvement. This data suggests that neurotrophic factor gene transfer using HSV-based vectors was able to reverse functional measures of established peripheral neuropathy in diabetic rodents and provides further evidence for the potential of HSV-based gene therapy to treat human diabetic neuropathy.
GENE THERAPY FOR CONNECTIVE TISSUE 672. The Effect of Serine Protease Inhibitor 6 (SPI-6) Gene Transfer on a Mouse Model of Sjögren’s Syndrome Fumi Mineshiba,1 Beatrijs M. Lodde,1 Jianghua Wang,1 Livia A. Casciola-Rosen,2 Antony Rosen,2 Bruce J. Baum.1 1 Gene Therapy and Therapeutics Branch, NIDCR-NIH-DHHS, Bethesda, MD; 2Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD. Sjögren’s syndrome (SS) is an autoimmune disease characterized by a focal and diffuse lymphoid cell infiltration into the salivary and lacrimal glands. This chronic immune cell activation leads to reduced secretory function with symptoms of xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). It has been hypothesized that these sequelae are due to apoptosis of the exocrine epithelial cells. Apoptosis can be induced by cytotoxic T-lymphocytes (CTLs), which contain the enzyme Granzyme B (GrB) in cytoplasmic granules. GrB initiates a caspase-independent apoptosis pathway and is inhibited by serine proteinase inhibitor 6 (SPI-6) in the mouse. In this study, we evaluated the effect of transgenic SPI-6 produced S259
670. Mutation Independent Silencing of Genes Involved in Retinal Degeneration by RNA Interference (RNAi) and Trans Complementation by a Codon Exchanged RNAi-Resistant Transgene Siobhan Cashman, Erin Binkley, Rajendra Kumar-Singh. 1 Ophthalmology and Human Genetics, University of Utah, Salt Lake City, UT. More than one hundred different mutations in the gene encoding rhodopsin are associated with a group of retinal degenerations including retinitis pigmentosa, congenital stationary night blindness and retinitis punctata albescens. Given this large heterogeneity of mutations, it would be ideal to develop mutation independent therapies for these diseases. We describe use of the endogenous RNA interference (RNAi) machinery present in eukaryotic cells to silence mutant and wild type rhodopsin alleles by a vector expressing a short hairpin RNA (shRNA). We found that in the human embryonic retinoblast cell line 911, wild type human rhodopsin driven by a CMV promoter could be silenced by 94.34±2.17% and the most common rhodopsin-associated mutation in the United States, P23H, could be silenced by 94.9±1.9% by a shRNA targeting rhodopsin. When the degeneracy of the genetic code was used to exchange 10 of 21 nucleotides of the region that encodes for the shRNA target in human rhodopsin cDNA, we found that the shRNA was no longer able to silence the codon exchanged rhodopsin mRNA (cmRNA). Moreover, the efficiency of translation of this cmRNA was equivalent to wild type rhodopsin mRNA. For proof of principle of treatment of autosomal dominant diseases using RNAi, we cotransfected mutant (P23H) rhodopsin cDNA with a rhodopsin cDNA engineered to code for a cmRNA and a shRNA into human embryonic retinoblast 911 cells and found specific silencing of the P23H rhodopsin by 90.64±5.19%. In addition, we present data on two alternative shRNA sequences targeting human rhodopsin, one of which also silences rhodopsin albeit less efficiently than the one described above. We have cloned each of these shRNA transcripts into AAV vectors and begun testing the potential of using this strategy in animal models of retinal degeneration. In conclusion, we have identified an extremely potent shRNA sequence and a strategy to evade shRNA mediated silencing of mRNA that has the potential to be used for the treatment of a very large variety of retinal degenerations in a mutation independent manner.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
671. Nerve Growth Factor and Neurotrophin-3 Delivered by HSV Gene Transfer Reverses Diabetic Neuropathy James R. Goss,1 Munmun Chattopadhyay,2 William F. Goins,1 Darren Wolfe,1 Marina Mata,2 Joseph C. Glorioso,1 David J. Fink.2 1 Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA; 2Neurology, University of Michigan, Ann Arbor, MI. Peripheral neuropathy, involving motor, sensory, and autonomic nerves, is a common and debilitating complication of diabetes. Many features of human diabetic neuropathy can be modeled in the mouse using streptozotocin (STZ) to selectively destroy pancreatic beta cells resulting in sustained hyperglycemia but not requiring insulin replacement. We have previously demonstrated that recombinant replication defective herpes simplex virus (HSV)-based vectors expressing the neurotrophic proteins NGF (vector SHN) or NT3 (vector QL2HNT3) delivered by subcutaneous inoculation to transduce dorsal root ganglia prevents the development of neuropathy in the STZ mouse. Using a double-blind experiment, we have extended these findings by examining if these vectors could reverse STZ-induced neuropathy once it has been established. Male Swiss-Webster mice were injected with streptozotocin (STZ) and six weeks later the presence of peripheral neuropathy was confirmed by electrophysiological measurements of a foot sensory nerve response to an evoked potential and the foot withdrawal latency from a heat source. STZ-treated mice were then randomized to four treatment groups: 1) No vector; 2) vector SHZ (control vector); 3) vector SHN; or, 4) vector QL2HNT3. Mice injected with STZ developed a peripheral neuropathy characterized by a decrease in the foot nerve sensory amplitude, an increase in the foot withdrawal latency from a heat source, and a decrease in the number of nerve fibers innervating the skin of the hind foot. These indices of neuropathy were reversed 5 weeks after mice were inoculated with either SHN or QL2HNT3 into the hind foot; mice inoculated with a control vector (SHZ) demonstrated no improvement. This data suggests that neurotrophic factor gene transfer using HSV-based vectors was able to reverse functional measures of established peripheral neuropathy in diabetic rodents and provides further evidence for the potential of HSV-based gene therapy to treat human diabetic neuropathy.
GENE THERAPY FOR CONNECTIVE TISSUE 672. The Effect of Serine Protease Inhibitor 6 (SPI-6) Gene Transfer on a Mouse Model of Sjögren’s Syndrome Fumi Mineshiba,1 Beatrijs M. Lodde,1 Jianghua Wang,1 Livia A. Casciola-Rosen,2 Antony Rosen,2 Bruce J. Baum.1 1 Gene Therapy and Therapeutics Branch, NIDCR-NIH-DHHS, Bethesda, MD; 2Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD. Sjögren’s syndrome (SS) is an autoimmune disease characterized by a focal and diffuse lymphoid cell infiltration into the salivary and lacrimal glands. This chronic immune cell activation leads to reduced secretory function with symptoms of xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). It has been hypothesized that these sequelae are due to apoptosis of the exocrine epithelial cells. Apoptosis can be induced by cytotoxic T-lymphocytes (CTLs), which contain the enzyme Granzyme B (GrB) in cytoplasmic granules. GrB initiates a caspase-independent apoptosis pathway and is inhibited by serine proteinase inhibitor 6 (SPI-6) in the mouse. In this study, we evaluated the effect of transgenic SPI-6 produced S259