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456. Multi-Subtype Targeting, Escape-Reducing (MuSTER) Anti-HIV sh RNA Gene Therapy
OLIGONUCLEOTIDE & RNAI THERAPEUTICS 456. Multi-Subtype Targeting, Escape-Reducing (MuSTER) Anti-HIV shRNA Gene Therapy
Ulrike Jung,1 John J. Rossi.1 1 Dept. of Molecular & Cell Biology, Beckman Research Institute of City of Hope, Duarte. According to the WHO in 2009, 34 million people were HIV infected and 1.8 million AIDS-related deaths reported. The current gold standard of treatment for HIV, a cocktail of drugs, prolongs the survival of patients. However, over time viral escape renders the drugs ineffective thus the treatment is now increasingly named “combination anti-retroviral therapy” (cART). Furthermore, cART is incapable of eradicating viral reservoirs, as the virus rebounds during cART cessation. RNA interference (RNAi) is a widely used tool for specific and efficient reduction of gene expression. The safety and feasibility have been demonstrated in clinical trials for genetic disorders, various cancers, and infectious diseases including HIV. However, the specificity of RNAi potentially limits the range of effectively targeted HIV-1 isolates and subtypes and escape mutants to RNAi therapeutics can evolve too. We have developed a more comprehensive RNAi approach, using RNAi triggers that can bind to closely related targets by virtue of non-Watson/Crick target base-pairing. Careful selection of conserved HIV-1 target sequences expands the range of subtypes susceptible to a single shRNA. Parallel use of the same approach to intercept predicted escape mutants is possible. Preliminary results against pNL4-3 HIV show significant activity despite non-Watson/Crick target base-pairing. Experiments with mutated strains and tests of long term kinetics are under way. We refer to this approach as Multi-Subtype Targeting and EscapeReducing (MuSTER) anti-HIV shRNA gene therapeutics.
457. RNA Interference-Based Gene Therapy for Parkinson’s Disease Using shRNA That Specifically Targeting the LRRK2 G2019S Allele
Liang Huang,1 Xiaomin Su,1 Juan Wang,1 Kathleen MaguireZeiss,1 Howard J. Federoff.1,2 1 Department of Neuroscience, Georgetown University Medical Center, Washington, DC; 2Department of Neurology, Georgetown University Medical Center, Washington, DC. Leucine-rich repeat kinase 2 (LRRK2) is a risk factor for Parkinson’s disease (PD). About 5-10% of the familial PD and 3% of the sporadic PD cases are associated with LRRK2 mutations. G2019S is the most common mutation in LRRK2. A single nucleotide substitution in the gene sequence results in a Glycine to Serine change within the kinase domain and increases LRRK2’s kinase activity, leading to a presumptive toxic, gain-of-function LRRK2. Herein, we developed an shRNA-based gene transfer reagent specifically targeting the LRRK2 G2019S allele, based on the work of Dr. Matthew Wood. We first tested the efficacy and specificity of the shRNA sequences with immortalized hybrid dopaminergic cells (MN9D) carrying inducible G2019S or wild type LRRK2. For MN9D-G2019S cells, a significant decrease in LRRK2 mRNA as well as protein levels was observed in cells expressing the G2019S specific shRNAp4 cells but not the scrambled shRNA (Scr shRNA). For MN9D-LRRK2 (wild type) cells, no significant changes in LRRK2 mRNA or protein levels were observed regardless of shRNA expression. Similar results were seen in MN9D-LRRK2 and –G2019S cells differentiated with n-butyrate. In neurite extension experiments, expression of shRNAp4 prevented the shortening of neurites caused by LRRK2 G2019S expression. To further test shRNAp4, we delivered the shRNA-expression constructs into human PD patient or normal fibroblasts. We found that shRNAp4 but not Scr shRNA decreased LRRK2 protein levels in PD patient fibroblasts but not normal fibroblasts. For in vivo studies, we are establishing an animal model with LRRK2 knockout rat that expresses wild type LRRK2 or LRRK2 G2019S. The shRNA expression S176
constructs will be delivered using recombinant adeno-associated virus 2 (rAAV2). In conclusion, our results show that shRNAp4 is able to allele specifically knock down LRRK2 G2019S and therefore is a candidate for RNA interference-based therapeutic approach to suppress LRRK2 G2019S-mediated dopaminergic neuron death. This work is supported by the Department of Defense.
458. An RNAi Screening Platform To Identify Modulators of Glioma Stem Cells Viability and Differentiation
Christian E. Badr,1,2 Romain Amante,1 Stephanie Van Hoppe,1 Bakhos A. Tannous.1,2 1 Massachusetts General Hospital, Boston; 2Harvard Medical School, Boston.
Glioma stem cells (GSCs) represent a subset population within malignant gliomas, one of the most aggressive brain tumors with very limited treatment options. These cells are highly tumorigenic, intrinsically resistant to current therapies, and capable of self-renewal and differentiation into mature glioma cells (GCs). Since the Glial Fibrillary Acidic Protein (GFAP) is highly expressed in mature GCs compared to GSCs, we have used its expression pattern as a differentiation marker and developed a bioluminescent reporter to monitor its activity. This reporter consists of a compact GFAP promoter driving the expression of the luciferase from the ostracod Vargula (Cypridina) hilgendorfii (Vluc; uses vargulin substrate) and the mCherry red fluorescent protein. On the other hand, a constitutively active promoter driving the expression of the Gaussia luciferase (uses coelenterazine as a substrate) and the cyan fluorescent protein is used to monitor cell viability. Since both luciferases are secreted, viability and differentiation of GSCs can be simultaneously monitored in real-time by collecting an aliquot of the cell-free conditioned medium (for in vitro assays) or blood (for in vivo assays) at different time points. Next we optimized a transfection protocol that allows delivering of RNAi to primary GSCs leading to efficient gene knockdown and subsequent changes in GSCs, which could be monitored using our reporter system in a high-throughput fashion. Using this bioluminescence-based platform for RNAi delivery and monitoring of GSCs, we are now able to perform a full genome RNAi screen to identify new GSCs modulators. Such screen is pivotal in the understanding of the biology of this elusive tumor population and in identifying new therapeutic gene targets.
459. Short Hairpin RNAs (shRNA) Against Steroid Receptor Coactivator-3/Amplified in Breast Cancer 1 (SRC-3) and Steroid Receptor Coactivator-1 (SRC-1) as Breast Cancer Therapeutic Agents
David M. Lonard,3 Donald Rao,2 John J. Nemunaitis,1,2 Neil N. Senzer,1,2 Bert W. O’Malley.3 1 Mary Crowley Cancer Research Centers, Dallas, TX; 2Gradalis, Inc., Carrollton, TX; 3Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX. Steroid receptor coactivator-3/amplified in breast cancer-1 (SRC-3/AIB-1) and steroid receptor coactivator-1 (SRC-1) are key breast cancer oncogenes that are frequently overexpressed or amplified in estrogen receptor and HER2 positive breast cancers. Experimental targeting of either SRC-3 or SRC-1 has been shown to limit breast cancer cell growth and restore the anti-estrogenic actions of tamoxifen. Here, are utilizing RNA interference (RNAi) technology with bifunctional shRNA (bi-shRNA)-based design for singlet SRC-1 or SRC-3 knockdown (with the capability for duplex construction). Bi-shRNA effectors achieve enhanced target knockdown by simultaneously promoting target mRNA cleavage, mRNA degradation (via p-body sequestration) and translational Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
Ulrike Jung,1 John J. Rossi.1 1 Dept. of Molecular & Cell Biology, Beckman Research Institute of City of Hope, Duarte. According to the WHO in 2009, 34 million people were HIV infected and 1.8 million AIDS-related deaths reported. The current gold standard of treatment for HIV, a cocktail of drugs, prolongs the survival of patients. However, over time viral escape renders the drugs ineffective thus the treatment is now increasingly named “combination anti-retroviral therapy” (cART). Furthermore, cART is incapable of eradicating viral reservoirs, as the virus rebounds during cART cessation. RNA interference (RNAi) is a widely used tool for specific and efficient reduction of gene expression. The safety and feasibility have been demonstrated in clinical trials for genetic disorders, various cancers, and infectious diseases including HIV. However, the specificity of RNAi potentially limits the range of effectively targeted HIV-1 isolates and subtypes and escape mutants to RNAi therapeutics can evolve too. We have developed a more comprehensive RNAi approach, using RNAi triggers that can bind to closely related targets by virtue of non-Watson/Crick target base-pairing. Careful selection of conserved HIV-1 target sequences expands the range of subtypes susceptible to a single shRNA. Parallel use of the same approach to intercept predicted escape mutants is possible. Preliminary results against pNL4-3 HIV show significant activity despite non-Watson/Crick target base-pairing. Experiments with mutated strains and tests of long term kinetics are under way. We refer to this approach as Multi-Subtype Targeting and EscapeReducing (MuSTER) anti-HIV shRNA gene therapeutics.
457. RNA Interference-Based Gene Therapy for Parkinson’s Disease Using shRNA That Specifically Targeting the LRRK2 G2019S Allele
Liang Huang,1 Xiaomin Su,1 Juan Wang,1 Kathleen MaguireZeiss,1 Howard J. Federoff.1,2 1 Department of Neuroscience, Georgetown University Medical Center, Washington, DC; 2Department of Neurology, Georgetown University Medical Center, Washington, DC. Leucine-rich repeat kinase 2 (LRRK2) is a risk factor for Parkinson’s disease (PD). About 5-10% of the familial PD and 3% of the sporadic PD cases are associated with LRRK2 mutations. G2019S is the most common mutation in LRRK2. A single nucleotide substitution in the gene sequence results in a Glycine to Serine change within the kinase domain and increases LRRK2’s kinase activity, leading to a presumptive toxic, gain-of-function LRRK2. Herein, we developed an shRNA-based gene transfer reagent specifically targeting the LRRK2 G2019S allele, based on the work of Dr. Matthew Wood. We first tested the efficacy and specificity of the shRNA sequences with immortalized hybrid dopaminergic cells (MN9D) carrying inducible G2019S or wild type LRRK2. For MN9D-G2019S cells, a significant decrease in LRRK2 mRNA as well as protein levels was observed in cells expressing the G2019S specific shRNAp4 cells but not the scrambled shRNA (Scr shRNA). For MN9D-LRRK2 (wild type) cells, no significant changes in LRRK2 mRNA or protein levels were observed regardless of shRNA expression. Similar results were seen in MN9D-LRRK2 and –G2019S cells differentiated with n-butyrate. In neurite extension experiments, expression of shRNAp4 prevented the shortening of neurites caused by LRRK2 G2019S expression. To further test shRNAp4, we delivered the shRNA-expression constructs into human PD patient or normal fibroblasts. We found that shRNAp4 but not Scr shRNA decreased LRRK2 protein levels in PD patient fibroblasts but not normal fibroblasts. For in vivo studies, we are establishing an animal model with LRRK2 knockout rat that expresses wild type LRRK2 or LRRK2 G2019S. The shRNA expression S176
constructs will be delivered using recombinant adeno-associated virus 2 (rAAV2). In conclusion, our results show that shRNAp4 is able to allele specifically knock down LRRK2 G2019S and therefore is a candidate for RNA interference-based therapeutic approach to suppress LRRK2 G2019S-mediated dopaminergic neuron death. This work is supported by the Department of Defense.
458. An RNAi Screening Platform To Identify Modulators of Glioma Stem Cells Viability and Differentiation
Christian E. Badr,1,2 Romain Amante,1 Stephanie Van Hoppe,1 Bakhos A. Tannous.1,2 1 Massachusetts General Hospital, Boston; 2Harvard Medical School, Boston.
Glioma stem cells (GSCs) represent a subset population within malignant gliomas, one of the most aggressive brain tumors with very limited treatment options. These cells are highly tumorigenic, intrinsically resistant to current therapies, and capable of self-renewal and differentiation into mature glioma cells (GCs). Since the Glial Fibrillary Acidic Protein (GFAP) is highly expressed in mature GCs compared to GSCs, we have used its expression pattern as a differentiation marker and developed a bioluminescent reporter to monitor its activity. This reporter consists of a compact GFAP promoter driving the expression of the luciferase from the ostracod Vargula (Cypridina) hilgendorfii (Vluc; uses vargulin substrate) and the mCherry red fluorescent protein. On the other hand, a constitutively active promoter driving the expression of the Gaussia luciferase (uses coelenterazine as a substrate) and the cyan fluorescent protein is used to monitor cell viability. Since both luciferases are secreted, viability and differentiation of GSCs can be simultaneously monitored in real-time by collecting an aliquot of the cell-free conditioned medium (for in vitro assays) or blood (for in vivo assays) at different time points. Next we optimized a transfection protocol that allows delivering of RNAi to primary GSCs leading to efficient gene knockdown and subsequent changes in GSCs, which could be monitored using our reporter system in a high-throughput fashion. Using this bioluminescence-based platform for RNAi delivery and monitoring of GSCs, we are now able to perform a full genome RNAi screen to identify new GSCs modulators. Such screen is pivotal in the understanding of the biology of this elusive tumor population and in identifying new therapeutic gene targets.
459. Short Hairpin RNAs (shRNA) Against Steroid Receptor Coactivator-3/Amplified in Breast Cancer 1 (SRC-3) and Steroid Receptor Coactivator-1 (SRC-1) as Breast Cancer Therapeutic Agents
David M. Lonard,3 Donald Rao,2 John J. Nemunaitis,1,2 Neil N. Senzer,1,2 Bert W. O’Malley.3 1 Mary Crowley Cancer Research Centers, Dallas, TX; 2Gradalis, Inc., Carrollton, TX; 3Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX. Steroid receptor coactivator-3/amplified in breast cancer-1 (SRC-3/AIB-1) and steroid receptor coactivator-1 (SRC-1) are key breast cancer oncogenes that are frequently overexpressed or amplified in estrogen receptor and HER2 positive breast cancers. Experimental targeting of either SRC-3 or SRC-1 has been shown to limit breast cancer cell growth and restore the anti-estrogenic actions of tamoxifen. Here, are utilizing RNA interference (RNAi) technology with bifunctional shRNA (bi-shRNA)-based design for singlet SRC-1 or SRC-3 knockdown (with the capability for duplex construction). Bi-shRNA effectors achieve enhanced target knockdown by simultaneously promoting target mRNA cleavage, mRNA degradation (via p-body sequestration) and translational Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy