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Micro RNAs as Novel siRNA Shuttles
CENTRAL NERVOUS SYSTEM I Methods: HAEC were cultured in media with low glucose (5.5 mM) or high glucose (30 mM). After 5 days, the cells with/without GTPCH gene transfer (cells alone and AdeGFP as controls) were prepared for assays of (1) NO with EPR; (2) SO with cytochrome C reduction and DHE; (3) BH4 with HPLC; (4) eNOS expression and dimerisation with immunoblotting (densitometric quantification of blots). All experiments were repeated twice (n = 6), and p value < 0.05 is considered to be significant. Results: HAEC in high glucose cultures compared with the low glucose had 30% decrease in NO and two-fold increase in SO, with reduced levels of total biopterin (7.2 vs 10.8) and BH4 (1.0 vs 2.9). eNOS expression in the high glucose was increased 1.5-fold, but mainly existed in the monomeric form (dimer:monomer ratio 0.3 vs 0.5). GTPCH gene transfer increased biopterins 30-40 fold as compared with controls. GTPCH gene transfer also increased NO production 1.6-fold, led to 30% SO reduction. Furthermore, augmented BH4 promoted eNOS dimerisation. The dimer:monomer ratio in GTPCH was 2.6-fold higher than in the controls. Conclusion: This study demonstrates a critical role for BH4 in regulating eNOS function in hyperglycaemic human endothelial cells. GTPCH gene transfer is a powerful approach to investigate eNOS regulation and potential therapeutic significance in diabetes.
CENTRAL NERVOUS SYSTEM I 207.
Micro RNAs as Novel siRNA Shuttles
Ryan Boudreau,1,2 Haibin Xia,1,2 Beverly L. Davidson.1,2,3,4 1 Program in Gene Therapy; 2Departments of Internal Medicine; 3 Neurology; 4Physiology & Biophysics, University of Iowa, Iowa City, IA. Huntington’s disease (HD) and Spinocerebellar ataxia type I (SCA1) are two of a class of dominant, neurodegenerative diseases caused by a polyglutamine (polyQ) expansion. The mutation confers a toxic gain of function to the protein, with polyQ length predictive of age of onset and disease severity. There is no curative or preventative therapy for HD or SCA1, supporting the investigation of novel strategies. In other work, we showed that gene silencing by RNA interference (RNAi) can be achieved in vitro and in vivo by expressing short hairpin RNAs (shRNAs) specific for mRNAs encoding ataxin-1 or huntingtin. Currently, strong, constitutive polIII promoters (U6 and H1) are used to express shRNAs, which are subsequently processed into functional small interfering RNAs (siRNAs). However, strong, constitutive expression of shRNAs may be inappropriate for diseases that take several decades to manifest. Moreover, high-level expression may be unnecessary for sustained benefit, and in some systems may induce a non-specific interferon response leading to global shut-down of gene expression. We therefore investigated the use of polII-expressed microRNAs (miRNAs) as siRNA shuttles as an alternative strategy. Due to their endogenous nature, miRNA backbones may prevent the induction of the interferon response. Using human mir-30 as a template, we designed miRNA shuttles that upon processing by dicer would release siRNAs specific for ataxin-1. Constructs containing polII-expressed miRNA shuttles with embedded ataxin1-specific siRNAs were co-transfected into cells with GFP-tagged ataxin-1, and gene silencing was assessed by fluorescence microscopy and western analysis. We observed dramatic and dose-dependent gene silencing relative to non-specific miRNAs carrying control siRNAs. This polII-based expression system exploits the structure of known miRNAs and supports tissue-specific as well as inducible siRNA expression, and thus, serves as a unique and powerful alternative to dominant neurodegenerative disease therapy by RNAi.
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208. Regenerative Properties of Retinoic Acid Receptor β2 in Rat Model of Dorsal Rhizotomy Liang-Fong Wong,1 Ping K. Yip,2 Anna Battaglia,2 John Grist,2 Lucy E. Walmsley,1 Malcolm Maden,3 Susan M. Kingsman,1 Alan J. Kingsman,1 Stephen B. McMahon,2 Nicholas D. Mazarakis.1 1 Neurobiology, Oxford Biomedica (UK) Ltd, Oxford, United Kingdom; 2Sensory Function Group, Centre for Neuroscience Research, King’s College London, London, United Kingdom; 3 MRC Centre for Developmental Biology, King’s College London, London, United Kingdom. After rhizotomy, axonal regeneration in the dorsal root is arrested in the transition zone between the peripheral and central nervous system. The damaged sensory axons have been shown to grow across this inhibitory dorsal root entry zone (DREZ) with the provision of neurotrophic factors1. In this study we present a novel approach to stimulating axonal growth across this barrier via the use of an equine infectious anaemia virus (EIAV)-based lentiviral vector expressing a novel regenerative molecule. Retinoic acid receptor β2 (RARβ2) is a transcription factor activated by retinoic acid, a biologically active metabolite of Vitamin A, and has been shown to induce neurite outgrowth in adult spinal cord explants2. An EIAVbased lentiviral vector expressing RARβ2 was delivered to sensory neurones via injection into dorsal spinal cord. In control animals that received an EIAV vector expressing β-galactosidase (LacZ), dorsal rhizotomy resulted in the loss of peripheral sensory axons innervating the spinal cord corresponding to the injured roots. In contrast, injured primary sensory afferents were found to regenerate across the DREZ in RARβ2 animals. Furthermore RARβ2-treated animals exhibited improvement in performance in behavioural tasks assessing for sensory and motor functions. These results suggest that lentiviral-mediated delivery of RARβ2 promoted the regeneration of injured sensory axons into the inhibitory DREZ after rhizotomy and promoted functional recovery. Hence this novel approach shows potential for therapeutic applications in the treatment of dorsal root avulsion injuries. 1 Ramer MS, Priestley JV, McMahon SB (2000) Functional regeneration of sensory axons into the adult spinal cord. Nature 403: 312-6 2 Corcoran J, So PL, Barber RD, Vincent KJ, Mazarakis ND, Mitrophanous KA, Kingsman SM, Maden M (2002) Retinoic acid receptor β2 and neurite outgrowth in the adult mouse spinal cord in vitro. J Cell Sci. 115: 3779-86
209. Gene Silencing of Human Huntingtin Using Lentivirus-Delivered shRNA Scott Q. Harper,1,2 Patrick D. Staber,1,2 Christine R. Rowley,1,2 Xiaohua He,1,2 Ines H. Martins,1,2 Qinwen Mao,1,2 Henry L. Paulson,3 Beverly L. Davidson.1,2,3,4 1 Program in Gene Therapy; 2Departments of Internal Medicine; 3 Neurology; 4Physiology & Biophysics, University of Iowa, Iowa City, IA. Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by polyglutamine expansion in huntingtin protein. Accumulation of mutant, toxic huntingtin in human patients and some HD mouse models leads to neurodegeneration, neurological impairment, and premature death. An effective treatment will require a reduction of mutant huntingtin. Induction of RNA interference (RNAi) by small hairpin RNA (shRNA) may be a feasible approach to suppress huntingtin gene expression and prevent its toxic accumulation in neurons. We hypothesize that stable expression of huntingtin-specific shRNA using feline immunodeficiency virus (FIV) vectors will provide longterm gene silencing, reduce mutant huntingtin expression and prevent or reverse HD-associated symptoms. Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy
CENTRAL NERVOUS SYSTEM I 207.
Micro RNAs as Novel siRNA Shuttles
Ryan Boudreau,1,2 Haibin Xia,1,2 Beverly L. Davidson.1,2,3,4 1 Program in Gene Therapy; 2Departments of Internal Medicine; 3 Neurology; 4Physiology & Biophysics, University of Iowa, Iowa City, IA. Huntington’s disease (HD) and Spinocerebellar ataxia type I (SCA1) are two of a class of dominant, neurodegenerative diseases caused by a polyglutamine (polyQ) expansion. The mutation confers a toxic gain of function to the protein, with polyQ length predictive of age of onset and disease severity. There is no curative or preventative therapy for HD or SCA1, supporting the investigation of novel strategies. In other work, we showed that gene silencing by RNA interference (RNAi) can be achieved in vitro and in vivo by expressing short hairpin RNAs (shRNAs) specific for mRNAs encoding ataxin-1 or huntingtin. Currently, strong, constitutive polIII promoters (U6 and H1) are used to express shRNAs, which are subsequently processed into functional small interfering RNAs (siRNAs). However, strong, constitutive expression of shRNAs may be inappropriate for diseases that take several decades to manifest. Moreover, high-level expression may be unnecessary for sustained benefit, and in some systems may induce a non-specific interferon response leading to global shut-down of gene expression. We therefore investigated the use of polII-expressed microRNAs (miRNAs) as siRNA shuttles as an alternative strategy. Due to their endogenous nature, miRNA backbones may prevent the induction of the interferon response. Using human mir-30 as a template, we designed miRNA shuttles that upon processing by dicer would release siRNAs specific for ataxin-1. Constructs containing polII-expressed miRNA shuttles with embedded ataxin1-specific siRNAs were co-transfected into cells with GFP-tagged ataxin-1, and gene silencing was assessed by fluorescence microscopy and western analysis. We observed dramatic and dose-dependent gene silencing relative to non-specific miRNAs carrying control siRNAs. This polII-based expression system exploits the structure of known miRNAs and supports tissue-specific as well as inducible siRNA expression, and thus, serves as a unique and powerful alternative to dominant neurodegenerative disease therapy by RNAi.
S80
208. Regenerative Properties of Retinoic Acid Receptor β2 in Rat Model of Dorsal Rhizotomy Liang-Fong Wong,1 Ping K. Yip,2 Anna Battaglia,2 John Grist,2 Lucy E. Walmsley,1 Malcolm Maden,3 Susan M. Kingsman,1 Alan J. Kingsman,1 Stephen B. McMahon,2 Nicholas D. Mazarakis.1 1 Neurobiology, Oxford Biomedica (UK) Ltd, Oxford, United Kingdom; 2Sensory Function Group, Centre for Neuroscience Research, King’s College London, London, United Kingdom; 3 MRC Centre for Developmental Biology, King’s College London, London, United Kingdom. After rhizotomy, axonal regeneration in the dorsal root is arrested in the transition zone between the peripheral and central nervous system. The damaged sensory axons have been shown to grow across this inhibitory dorsal root entry zone (DREZ) with the provision of neurotrophic factors1. In this study we present a novel approach to stimulating axonal growth across this barrier via the use of an equine infectious anaemia virus (EIAV)-based lentiviral vector expressing a novel regenerative molecule. Retinoic acid receptor β2 (RARβ2) is a transcription factor activated by retinoic acid, a biologically active metabolite of Vitamin A, and has been shown to induce neurite outgrowth in adult spinal cord explants2. An EIAVbased lentiviral vector expressing RARβ2 was delivered to sensory neurones via injection into dorsal spinal cord. In control animals that received an EIAV vector expressing β-galactosidase (LacZ), dorsal rhizotomy resulted in the loss of peripheral sensory axons innervating the spinal cord corresponding to the injured roots. In contrast, injured primary sensory afferents were found to regenerate across the DREZ in RARβ2 animals. Furthermore RARβ2-treated animals exhibited improvement in performance in behavioural tasks assessing for sensory and motor functions. These results suggest that lentiviral-mediated delivery of RARβ2 promoted the regeneration of injured sensory axons into the inhibitory DREZ after rhizotomy and promoted functional recovery. Hence this novel approach shows potential for therapeutic applications in the treatment of dorsal root avulsion injuries. 1 Ramer MS, Priestley JV, McMahon SB (2000) Functional regeneration of sensory axons into the adult spinal cord. Nature 403: 312-6 2 Corcoran J, So PL, Barber RD, Vincent KJ, Mazarakis ND, Mitrophanous KA, Kingsman SM, Maden M (2002) Retinoic acid receptor β2 and neurite outgrowth in the adult mouse spinal cord in vitro. J Cell Sci. 115: 3779-86
209. Gene Silencing of Human Huntingtin Using Lentivirus-Delivered shRNA Scott Q. Harper,1,2 Patrick D. Staber,1,2 Christine R. Rowley,1,2 Xiaohua He,1,2 Ines H. Martins,1,2 Qinwen Mao,1,2 Henry L. Paulson,3 Beverly L. Davidson.1,2,3,4 1 Program in Gene Therapy; 2Departments of Internal Medicine; 3 Neurology; 4Physiology & Biophysics, University of Iowa, Iowa City, IA. Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by polyglutamine expansion in huntingtin protein. Accumulation of mutant, toxic huntingtin in human patients and some HD mouse models leads to neurodegeneration, neurological impairment, and premature death. An effective treatment will require a reduction of mutant huntingtin. Induction of RNA interference (RNAi) by small hairpin RNA (shRNA) may be a feasible approach to suppress huntingtin gene expression and prevent its toxic accumulation in neurons. We hypothesize that stable expression of huntingtin-specific shRNA using feline immunodeficiency virus (FIV) vectors will provide longterm gene silencing, reduce mutant huntingtin expression and prevent or reverse HD-associated symptoms. Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy