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607. 2’F-Modified RNA Aptamers as Selective Inhibitors of N-Methyl D-Aspartate Receptors
NEUROLOGICAL DISEASES II 607. 2’F-Modified RNA Aptamers as Selective Inhibitors of N-Methyl D-Aspartate Receptors
Garam Lee,1 David M. MacLean,1 Henning Ulrich,2 Xiurong Zhao,3 Jaroslaw Aronowski,3 Vasanthi Jayaraman.1 1 Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, Houston, TX; 2 Department of Biochemistry, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil; 3Department of Neurology, University of Texas Health Science Center at Houston, Houston, TX. Excessive activation of N-methyl D-aspartate receptors (NMDARs) contributes to a wide range of neurological disorders including stroke, Alzheimer’s disease, Huntington’s disease and CNS trauma. NMDARs antagonists show potential in the treatment of stroke, but the inability for most antagonists to cross the blood-brain barrier hampers clinical trials. To address this, we are developing a watersoluble RNA molecule based antagonist selective for NMDARs. To do this, we used Systematic Evolution of Ligands by Exponential Enrichment (SELEX) for isolating NMDARs specific 2’F-RNA aptamers. SELEX is an in-vitro process of repetitive selection for isolating nucleic acid ligands that bind to targets of interest. By alternating positive selection rounds, to increase the number of RNA ligands which bind to the target, and negative selection rounds, to eliminate background bound RNA, a small number of specific high affinity ligands can be isolated. We used glutamate-displacement strategy on NR1-NR2A membrane protein as a positive selection with negative binding experiments of the AMPA and kainate receptors to arrive at a pool of RNA ligands selective for the NMDAR. Using outside-out patch clamp electrophysiology to test the RNA pools, we found that the 9th RNA pool was enriched with RNA antagonists of the NMDAR. The RNAs in this pool were cloned and 9 of 24 clones were found to have a 5-nt consensus motif. Subsequent patch clamp experiments found that best candidate strongly inhibit NMDAR activation, but weakly or partially block AMPA and kainate receptors. Binding assays on C26 (90b) RNA from this pool yield a Kd of 67 ± 3 nM at the NMDAR. The minimization (50 bp) of C26 contain functional motif was done based on its secondary prediction by Mfold. C26-50 RNAs show significant reduction of neurotoxicity in OGD assay. Based on patch-clamp, radio-ligand binding experiments and OGD assay, C26-50 RNA aptamer is a potent and selective antagonist for NMDARs.
608. Mutant HumanT8993G ATP6 Expression by a Mitochondrial Targeting Sequence Modification of AAV Capsid VP2 in Transgenic Mice Huijun Yuan,1 John Guy.1 1 Ophthamology, Bascom Palmer Eye Institute, University of Miami, Miami, FL.
Purpose: To create a mouse model of human mutant ATP6, at T8993G of mitochondrial DNA by a mitochondrial targeting sequence modification of AAV capsid VP2. The ATP6 mutation causes two mitochondrial diseases, Neuropathy, ataxia, retinitis pigmentosa (NARP) and Maternally-inherited Leigh’s syndrome (MILS). Methods: Sc-HSP-ATP6mut fused with FLAG and a second gene mitochondrial mCherry was packaged with mito-targeted (COX8) VP2 capsid into AAV2. ATP6mut (A6M) transgenic mice were created with injections of AAV2-cox8ATP6mutFlag-mcherry into fertilized eggs. The Heidelberg image was used to examine mCherry expression of the A6M mice at two months. The expression of ATP6mutFLAGcherry was detected by immunohistochemistry and blue native PAGE gel with 2D western blotting. The brain degeneration of ATP6 mutant mice were checked by MRI and H&E staining. The pattern electroretinogram (PERG) and flash ERG examined visual function. Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Results: Using Heidelberg imaging, mCherry was detected by red channels in 80% of the A6M transgenic mice. Two A6M mice died at one month and three others died between 2 to 5 months. MCherry and Flag tagged human ATP6 mutant gene exhibited a perinuclear distribution and colocalization to the mitochondrial marker porin in the brain, spinal cord, lung, heart and muscles. A blue native PAGE and 2D SDS-PAGE western blotting showed human mutant ATP6 expression was detected in complex V by anti-flag. MRI revealed significant, symmetrical lesions observed in the brains of A6M mice. Histopathology of dead, mice brains displayed demyelination in the cerebellum. At 4 months, PERG data showed amplitudes decreased in A6M mice to 10 uV compared to 31 uV in wild-type mice (P=0.0001). Conclusions: A modified AAV VP2 with a mitochondrial targeting sequencing can successfully deliver a human ATP6mutant gene to mouse mitochondria, similar to human mitochondrial diseases, NARP and MILS. This mouse model can be used to test therapies for these blinding and fatal disorders.
609. Development of Novel DNA Vaccines for the Prevention of Botulinum Neurotoxin Poisoning
Veronica L. Scott,1 Natalie A. Hutnick,1 Daniel Villarreal,1 Edwin Ragwan,1 David B. Weiner.1 1 Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA.
Botulinum neurotoxins (BoNTs) are some of the most deadly toxic substances known to humankind. Botulinum neurotoxin intoxication results in a severe and potentially fatal neurological disease characterized by acute flaccid paralysis of motor and autonomic nerves. Although the Center for Disease Control (CDC) has identified the use of botulinum toxin as a potential bioterrorist weapon, there is currently no licensed vaccine to prevent botulinum intoxication. Here we present studies of a novel vaccine developed using an optimized DNA platform with the goal to produce broad immunity against the lethal Clostridium botulinum neurotoxin serotypes (A, B, and E). Single-plasmid vector systems expressing the non-toxic heavy chains (Hcs) of the neurotoxins were constructed. Optimal dosages of the BoNT vaccines were determined by performing dose escalation studies in Balb/c mice. The protective efficacies of the vaccines were determined by performing challenge studies with 102 LD50 of each respective neurotoxin. Low dose vaccination (10 μg) with each of the monovalent vaccines (A, B, or E) and with the trivalent A, B, and E cocktail vaccine induced robust humoral and polyfunctional CD4+ T-cell responses and provided 100% protection against lethal challenge with the respective Clostridium botulinum neurotoxin in mice. Additionally, as the correlate of protection for botulinum intoxication is antibody mediated, we evaluated whether antibodies induced by the monovalent vaccines were capable of protecting naïve animals from challenge. We observed that naïve animals administered a 1:1 ratio of immunized sera with 102 LD50 of each respective neurotoxin were 100% protected against intoxication. The data demonstrate the protective efficacy induced by monovalent and trivalent DNA vaccines targeting the botulinum serotypes (A, B, and E) responsible for human intoxication. This study has importance for the development of vaccines that provide protective immunity against potential bioterrorist toxins.
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Garam Lee,1 David M. MacLean,1 Henning Ulrich,2 Xiurong Zhao,3 Jaroslaw Aronowski,3 Vasanthi Jayaraman.1 1 Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, Houston, TX; 2 Department of Biochemistry, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil; 3Department of Neurology, University of Texas Health Science Center at Houston, Houston, TX. Excessive activation of N-methyl D-aspartate receptors (NMDARs) contributes to a wide range of neurological disorders including stroke, Alzheimer’s disease, Huntington’s disease and CNS trauma. NMDARs antagonists show potential in the treatment of stroke, but the inability for most antagonists to cross the blood-brain barrier hampers clinical trials. To address this, we are developing a watersoluble RNA molecule based antagonist selective for NMDARs. To do this, we used Systematic Evolution of Ligands by Exponential Enrichment (SELEX) for isolating NMDARs specific 2’F-RNA aptamers. SELEX is an in-vitro process of repetitive selection for isolating nucleic acid ligands that bind to targets of interest. By alternating positive selection rounds, to increase the number of RNA ligands which bind to the target, and negative selection rounds, to eliminate background bound RNA, a small number of specific high affinity ligands can be isolated. We used glutamate-displacement strategy on NR1-NR2A membrane protein as a positive selection with negative binding experiments of the AMPA and kainate receptors to arrive at a pool of RNA ligands selective for the NMDAR. Using outside-out patch clamp electrophysiology to test the RNA pools, we found that the 9th RNA pool was enriched with RNA antagonists of the NMDAR. The RNAs in this pool were cloned and 9 of 24 clones were found to have a 5-nt consensus motif. Subsequent patch clamp experiments found that best candidate strongly inhibit NMDAR activation, but weakly or partially block AMPA and kainate receptors. Binding assays on C26 (90b) RNA from this pool yield a Kd of 67 ± 3 nM at the NMDAR. The minimization (50 bp) of C26 contain functional motif was done based on its secondary prediction by Mfold. C26-50 RNAs show significant reduction of neurotoxicity in OGD assay. Based on patch-clamp, radio-ligand binding experiments and OGD assay, C26-50 RNA aptamer is a potent and selective antagonist for NMDARs.
608. Mutant HumanT8993G ATP6 Expression by a Mitochondrial Targeting Sequence Modification of AAV Capsid VP2 in Transgenic Mice Huijun Yuan,1 John Guy.1 1 Ophthamology, Bascom Palmer Eye Institute, University of Miami, Miami, FL.
Purpose: To create a mouse model of human mutant ATP6, at T8993G of mitochondrial DNA by a mitochondrial targeting sequence modification of AAV capsid VP2. The ATP6 mutation causes two mitochondrial diseases, Neuropathy, ataxia, retinitis pigmentosa (NARP) and Maternally-inherited Leigh’s syndrome (MILS). Methods: Sc-HSP-ATP6mut fused with FLAG and a second gene mitochondrial mCherry was packaged with mito-targeted (COX8) VP2 capsid into AAV2. ATP6mut (A6M) transgenic mice were created with injections of AAV2-cox8ATP6mutFlag-mcherry into fertilized eggs. The Heidelberg image was used to examine mCherry expression of the A6M mice at two months. The expression of ATP6mutFLAGcherry was detected by immunohistochemistry and blue native PAGE gel with 2D western blotting. The brain degeneration of ATP6 mutant mice were checked by MRI and H&E staining. The pattern electroretinogram (PERG) and flash ERG examined visual function. Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Results: Using Heidelberg imaging, mCherry was detected by red channels in 80% of the A6M transgenic mice. Two A6M mice died at one month and three others died between 2 to 5 months. MCherry and Flag tagged human ATP6 mutant gene exhibited a perinuclear distribution and colocalization to the mitochondrial marker porin in the brain, spinal cord, lung, heart and muscles. A blue native PAGE and 2D SDS-PAGE western blotting showed human mutant ATP6 expression was detected in complex V by anti-flag. MRI revealed significant, symmetrical lesions observed in the brains of A6M mice. Histopathology of dead, mice brains displayed demyelination in the cerebellum. At 4 months, PERG data showed amplitudes decreased in A6M mice to 10 uV compared to 31 uV in wild-type mice (P=0.0001). Conclusions: A modified AAV VP2 with a mitochondrial targeting sequencing can successfully deliver a human ATP6mutant gene to mouse mitochondria, similar to human mitochondrial diseases, NARP and MILS. This mouse model can be used to test therapies for these blinding and fatal disorders.
609. Development of Novel DNA Vaccines for the Prevention of Botulinum Neurotoxin Poisoning
Veronica L. Scott,1 Natalie A. Hutnick,1 Daniel Villarreal,1 Edwin Ragwan,1 David B. Weiner.1 1 Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA.
Botulinum neurotoxins (BoNTs) are some of the most deadly toxic substances known to humankind. Botulinum neurotoxin intoxication results in a severe and potentially fatal neurological disease characterized by acute flaccid paralysis of motor and autonomic nerves. Although the Center for Disease Control (CDC) has identified the use of botulinum toxin as a potential bioterrorist weapon, there is currently no licensed vaccine to prevent botulinum intoxication. Here we present studies of a novel vaccine developed using an optimized DNA platform with the goal to produce broad immunity against the lethal Clostridium botulinum neurotoxin serotypes (A, B, and E). Single-plasmid vector systems expressing the non-toxic heavy chains (Hcs) of the neurotoxins were constructed. Optimal dosages of the BoNT vaccines were determined by performing dose escalation studies in Balb/c mice. The protective efficacies of the vaccines were determined by performing challenge studies with 102 LD50 of each respective neurotoxin. Low dose vaccination (10 μg) with each of the monovalent vaccines (A, B, or E) and with the trivalent A, B, and E cocktail vaccine induced robust humoral and polyfunctional CD4+ T-cell responses and provided 100% protection against lethal challenge with the respective Clostridium botulinum neurotoxin in mice. Additionally, as the correlate of protection for botulinum intoxication is antibody mediated, we evaluated whether antibodies induced by the monovalent vaccines were capable of protecting naïve animals from challenge. We observed that naïve animals administered a 1:1 ratio of immunized sera with 102 LD50 of each respective neurotoxin were 100% protected against intoxication. The data demonstrate the protective efficacy induced by monovalent and trivalent DNA vaccines targeting the botulinum serotypes (A, B, and E) responsible for human intoxication. This study has importance for the development of vaccines that provide protective immunity against potential bioterrorist toxins.
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