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Chen L, Bosmajian C, Woo S. Mechanistic intracellular PK/PD modeling to inform development strategies for small interfering RNA therapeutics. MOLECULAR THERAPY. NUCLEIC ACIDS 2025; 36:102516. [PMID: 40242045 PMCID: PMC12002994 DOI: 10.1016/j.omtn.2025.102516] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/05/2024] [Accepted: 03/12/2025] [Indexed: 04/18/2025]
Abstract
Small interfering RNA (siRNA) therapeutics provide a targeted approach to silence disease-related genes, with notable success in liver-targeting applications. However, the quantitative effects of siRNA properties, such as stability and affinity, as well as biological factors like cell proliferation, mRNA turnover, and abundance, on gene silencing, particularly for extrahepatic targets, remain poorly understood. To identify determinants influencing gene knockdown extent and duration, we developed a mechanistic intracellular pharmacokinetic/pharmacodynamic (PK/PD) model for RNAiMAX-delivered siRNA, based on cytoplasmic siRNA disposition, RISC-loaded siRNA exposure, and mRNA knockdown across different targets in MCF7 and BT474 cells. The model highlighted the critical roles of cell proliferation in silencing duration and mRNA turnover rates on knockdown extent. In rapid-dividing cells, mRNA half-life drives knockdown profiles, whereas chemical siRNA stabilization extends silencing in slow-dividing cells. Targets with extremely low or high mRNA abundance pose silencing challenges. While sufficient RISC occupancy is essential, increasing RISC exposure has minimal impact on silencing extent; enhancing siRNA-mRNA target engagement is more effective. The model also defined a quantitative relationship for maximal mRNA knockdown, governed by cell proliferation, mRNA half-life, and RISC-mediated cleavage rates. This mechanistic PK/PD modeling provides insights into optimizing siRNA design and target selection in therapeutic development.
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Affiliation(s)
- Lin Chen
- Division of Pharmacokinetics-Pharmacodynamics and Systems Pharmacology, Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14214, USA
| | - Caroline Bosmajian
- Division of Pharmacokinetics-Pharmacodynamics and Systems Pharmacology, Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14214, USA
| | - Sukyung Woo
- Division of Pharmacokinetics-Pharmacodynamics and Systems Pharmacology, Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14214, USA
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2
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Singh J, Saeedan AS, Kaithwas G, Ansari MN. Small interfering RNA: From designing to therapeutic in cancer. J Genet Eng Biotechnol 2025; 23:100484. [PMID: 40390497 PMCID: PMC11999615 DOI: 10.1016/j.jgeb.2025.100484] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2025] [Revised: 03/13/2025] [Accepted: 03/23/2025] [Indexed: 05/21/2025]
Abstract
Cancer has become a significant public health concern worldwide. It is a group of diseases, often resulting from the dysregulation of multiple cellular pathways involved in differentiation, cell proliferation, cell cycle regulation, and DNA repair. These disruptions are primarily caused by genetic mutation and epigenetic alterations which lead to uncontrolled growth and tumor formation. Targeted therapy is a precise and effective strategy to overcome the shortcomings of conventional therapy. RNA interference (RNAi) is a gene-silencing mechanism that has an uncanny ability to target disease-associated genes. Small interfering RNA (siRNA) is a key component of RNAi and has shown promise in silencing oncogenes and inhibiting cancer progression. However, the therapeutic application of siRNA faces several challenges such as poor cellular uptake, short half-life, endosomal escape, immune system activation, and off-target. Strategies to address these challenges are optimized designing of siRNA, advanced delivery systems, and chemical modification to improve cellular uptake and protect from degradation. This review focuses on the therapeutic potential of siRNA in cancer treatment and discusses the action mechanism of siRNA, barriers in siRNA, and strategies to overcome them. The review shed light on the current clinical trial of siRNA-based cancer therapy, along with outcomes and limitations.
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Affiliation(s)
- Jyoti Singh
- Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University (A Central University), Vidya Vihar, Raebareli Road, Lucknow 226025 Uttar Pradesh, India
| | - Abdulaziz S Saeedan
- Department of Pharmacology and Toxicology, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Alkharj 11942, Saudi Arabia
| | - Gaurav Kaithwas
- Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University (A Central University), Vidya Vihar, Raebareli Road, Lucknow 226025 Uttar Pradesh, India
| | - Mohd Nazam Ansari
- Department of Pharmacology and Toxicology, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Alkharj 11942, Saudi Arabia.
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3
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Supe S, Dighe V, Upadhya A, Singh K. Analysis of RNA Interference Targeted Against Human Antigen R (HuR) to Reduce Vascular Endothelial Growth Factor (VEGF) Protein Expression in Human Retinal Pigment Epithelial Cells. Mol Biotechnol 2024; 66:2972-2984. [PMID: 37856012 DOI: 10.1007/s12033-023-00913-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2023] [Accepted: 09/18/2023] [Indexed: 10/20/2023]
Abstract
VEGF-A or vascular endothelial growth factor-A is an important factor in enabling neovascularization and angiogenesis. VEGF-A is regulated transcriptionally as well as post transcriptionally. Human antigen R (HuR) belonging to the embryonic lethal abnormal vision (ELAV) family is a key regulator promoting stabilization of VEGF-A mRNA. In this research we investigate, whether HuR targeted RNA interference would enable the reduction of the VEGF-A protein in human retinal pigment epithelial cells (ARPE-19) in-vitro, in normoxic conditions. Three siRNA molecules with sequences complementary to three regions of the HuR mRNA were designed. The three designed siRNA molecules were individually transfected in ARPE-19 cells using Lipofectamine™2000 reagent. Post-transfection (24 h, 48 h, 72 h), downregulation of HuR mRNA was estimated by real-time polymerase reaction, while HuR protein and VEGF-A protein levels were semi-quantitatively determined by western blotting techniques. VEGF-A protein levels were additionally quantified using ELISA techniques. All experiments were done in triplicate. The designed siRNA could successfully downregulate HuR mRNA with concomitant decreases in HuR and VEGF-A protein. The study reveals that HuR downregulation can prominently downregulate VEGF-A, making the protein a target for therapy against pathological angiogenesis conditions such as diabetic retinopathy.
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Affiliation(s)
- Shibani Supe
- Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM'S NMIMS, Vile Parle (W), Mumbai, Maharashtra, 400056, India
| | - Vikas Dighe
- National Centre for Preclinical Reproductive and Genetic Toxicology, ICMR-National Institute for Research in Reproductive and Child Health, J.M. Street, Parel, Mumbai, Maharashtra, 400012, India
| | - Archana Upadhya
- Maharashtra Educational Society's H. K. College of Pharmacy, H. K. College Campus, Oshiwara, Jogeshwari (W), Mumbai, Maharashtra, 400102, India.
| | - Kavita Singh
- Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM'S NMIMS, Vile Parle (W), Mumbai, Maharashtra, 400056, India.
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4
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Liu C, Kogel K, Ladera‐Carmona M. Harnessing RNA interference for the control of Fusarium species: A critical review. MOLECULAR PLANT PATHOLOGY 2024; 25:e70011. [PMID: 39363756 PMCID: PMC11450251 DOI: 10.1111/mpp.70011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/14/2024] [Revised: 08/13/2024] [Accepted: 09/10/2024] [Indexed: 10/05/2024]
Abstract
Fusarium fungi are a pervasive threat to global agricultural productivity. They cause a spectrum of plant diseases that result in significant yield losses and threaten food safety by producing mycotoxins that are harmful to human and animal health. In recent years, the exploitation of the RNA interference (RNAi) mechanism has emerged as a promising avenue for the control of Fusarium-induced diseases, providing both a mechanistic understanding of Fusarium gene function and a potential strategy for environmentally sustainable disease management. However, despite significant progress in elucidating the presence and function of the RNAi pathway in different Fusarium species, a comprehensive understanding of its individual protein components and underlying silencing mechanisms remains elusive. Accordingly, while a considerable number of RNAi-based approaches to Fusarium control have been developed and many reports of RNAi applications in Fusarium control under laboratory conditions have been published, the applicability of this knowledge in agronomic settings remains an open question, and few convincing data on RNAi-based disease control under field conditions have been published. This review aims to consolidate the current knowledge on the role of RNAi in Fusarium disease control by evaluating current research and highlighting important avenues for future investigation.
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Affiliation(s)
- Caihong Liu
- Institute of Phytopathology, Research Centre for BioSystems, Land Use and NutritionJustus Liebig University GiessenGiessenGermany
| | - Karl‐Heinz Kogel
- Institute of Phytopathology, Research Centre for BioSystems, Land Use and NutritionJustus Liebig University GiessenGiessenGermany
- Institut de Biologie Moléculaire des Plantes, CNRSUniversité de StrasbourgStrasbourgFrance
| | - Maria Ladera‐Carmona
- Institute of Phytopathology, Research Centre for BioSystems, Land Use and NutritionJustus Liebig University GiessenGiessenGermany
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Sarli SL, Fakih HH, Kelly K, Devi G, Rembetsy-Brown J, McEachern H, Ferguson C, Echeverria D, Lee J, Sousa J, Sleiman H, Khvorova A, Watts J. Quantifying the activity profile of ASO and siRNA conjugates in glioblastoma xenograft tumors in vivo. Nucleic Acids Res 2024; 52:4799-4817. [PMID: 38613388 PMCID: PMC11109979 DOI: 10.1093/nar/gkae260] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2024] [Revised: 03/06/2024] [Accepted: 03/27/2024] [Indexed: 04/14/2024] Open
Abstract
Glioblastoma multiforme is a universally lethal brain tumor that largely resists current surgical and drug interventions. Despite important advancements in understanding GBM biology, the invasiveness and heterogeneity of these tumors has made it challenging to develop effective therapies. Therapeutic oligonucleotides-antisense oligonucleotides and small-interfering RNAs-are chemically modified nucleic acids that can silence gene expression in the brain. However, activity of these oligonucleotides in brain tumors remains inadequately characterized. In this study, we developed a quantitative method to differentiate oligonucleotide-induced gene silencing in orthotopic GBM xenografts from gene silencing in normal brain tissue, and used this method to test the differential silencing activity of a chemically diverse panel of oligonucleotides. We show that oligonucleotides chemically optimized for pharmacological activity in normal brain tissue do not show consistent activity in GBM xenografts. We then survey multiple advanced oligonucleotide chemistries for their activity in GBM xenografts. Attaching lipid conjugates to oligonucleotides improves silencing in GBM cells across several different lipid classes. Highly hydrophobic lipid conjugates cholesterol and docosanoic acid enhance silencing but at the cost of higher neurotoxicity. Moderately hydrophobic, unsaturated fatty acid and amphiphilic lipid conjugates still improve activity without compromising safety. These oligonucleotide conjugates show promise for treating glioblastoma.
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Affiliation(s)
- Samantha L Sarli
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Hassan H Fakih
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Karen Kelly
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Gitali Devi
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Julia M Rembetsy-Brown
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Holly R McEachern
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Chantal M Ferguson
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Dimas Echeverria
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Jonathan Lee
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Jacquelyn Sousa
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Hanadi F Sleiman
- Department of Chemistry, McGill University, Montréal, Québec, Canada
| | - Anastasia Khvorova
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Jonathan K Watts
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA
- Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA, USA
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6
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Buthelezi LA, Pillay S, Ntuli NN, Gcanga L, Guler R. Antisense Therapy for Infectious Diseases. Cells 2023; 12:2119. [PMID: 37626929 PMCID: PMC10453568 DOI: 10.3390/cells12162119] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2023] [Revised: 08/15/2023] [Accepted: 08/17/2023] [Indexed: 08/27/2023] Open
Abstract
Infectious diseases, particularly Tuberculosis (TB) caused by Mycobacterium tuberculosis, pose a significant global health challenge, with 1.6 million reported deaths in 2021, making it the most fatal disease caused by a single infectious agent. The rise of drug-resistant infectious diseases adds to the urgency of finding effective and safe intervention therapies. Antisense therapy uses antisense oligonucleotides (ASOs) that are short, chemically modified, single-stranded deoxyribonucleotide molecules complementary to their mRNA target. Due to their designed target specificity and inhibition of a disease-causing gene at the mRNA level, antisense therapy has gained interest as a potential therapeutic approach. This type of therapy is currently utilized in numerous diseases, such as cancer and genetic disorders. Currently, there are limited but steadily increasing studies available that report on the use of ASOs as treatment for infectious diseases. This review explores the sustainability of FDA-approved and preclinically tested ASOs as a treatment for infectious diseases and the adaptability of ASOs for chemical modifications resulting in reduced side effects with improved drug delivery; thus, highlighting the potential therapeutic uses of ASOs for treating infectious diseases.
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Affiliation(s)
- Lwanda Abonga Buthelezi
- International Centre for Genetic Engineering and Biotechnology, Cape Town Component, Cape Town 7925, South Africa; (L.A.B.); (S.P.); (N.N.N.); (L.G.)
- Department of Pathology, Division of Immunology, Institute of Infectious Diseases and Molecular Medicine (IDM), Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa
| | - Shandre Pillay
- International Centre for Genetic Engineering and Biotechnology, Cape Town Component, Cape Town 7925, South Africa; (L.A.B.); (S.P.); (N.N.N.); (L.G.)
- Department of Pathology, Division of Immunology, Institute of Infectious Diseases and Molecular Medicine (IDM), Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa
| | - Noxolo Nokukhanya Ntuli
- International Centre for Genetic Engineering and Biotechnology, Cape Town Component, Cape Town 7925, South Africa; (L.A.B.); (S.P.); (N.N.N.); (L.G.)
- Department of Pathology, Division of Immunology, Institute of Infectious Diseases and Molecular Medicine (IDM), Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa
| | - Lorna Gcanga
- International Centre for Genetic Engineering and Biotechnology, Cape Town Component, Cape Town 7925, South Africa; (L.A.B.); (S.P.); (N.N.N.); (L.G.)
- Department of Pathology, Division of Immunology, Institute of Infectious Diseases and Molecular Medicine (IDM), Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa
| | - Reto Guler
- International Centre for Genetic Engineering and Biotechnology, Cape Town Component, Cape Town 7925, South Africa; (L.A.B.); (S.P.); (N.N.N.); (L.G.)
- Department of Pathology, Division of Immunology, Institute of Infectious Diseases and Molecular Medicine (IDM), Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa
- Faculty of Health Sciences, Wellcome Centre for Infectious Diseases Research in Africa, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town 7925, South Africa
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7
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Thakur S, Sinhari A, Jain P, Jadhav HR. A perspective on oligonucleotide therapy: Approaches to patient customization. Front Pharmacol 2022; 13:1006304. [PMID: 36339619 PMCID: PMC9626821 DOI: 10.3389/fphar.2022.1006304] [Citation(s) in RCA: 58] [Impact Index Per Article: 19.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2022] [Accepted: 10/05/2022] [Indexed: 09/12/2023] Open
Abstract
It is estimated that the human genome encodes 15% of proteins that are considered to be disease-modifying. Only 2% of these proteins possess a druggable site that the approved clinical candidates target. Due to this disparity, there is an immense need to develop therapeutics that may better mitigate the disease or disorders aroused by non-druggable and druggable proteins or enzymes. The recent surge in approved oligonucleotide therapeutics (OT) indicates the imminent potential of these therapies. Oligonucleotide-based therapeutics are of intermediate size with much-improved selectivity towards the target and fewer off-target effects than small molecules. The OTs include Antisense RNAs, MicroRNA (MIR), small interfering RNA (siRNA), and aptamers, which are currently being explored for their use in neurodegenerative disorders, cancer, and even orphan diseases. The present review is a congregated effort to present the past and present of OTs and the current efforts to make OTs for plausible future therapeutics. The review provides updated literature on the challenges and bottlenecks of OT and recent advancements in OT drug delivery. Further, this review deliberates on a newly emerging approach to personalized treatment for patients with rare and fatal diseases with OT.
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Affiliation(s)
- Shikha Thakur
- Pharmaceutical Chemistry Laboratory, Department of Pharmacy, Birla Institute of Technology and Sciences Pilani, Pilani, RJ, India
| | - Apurba Sinhari
- Pharmaceutical Chemistry Laboratory, Department of Pharmacy, Birla Institute of Technology and Sciences Pilani, Pilani, RJ, India
| | - Priti Jain
- Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Delhi Pharmaceutical Sciences and Research University, New Delhi, India
| | - Hemant R. Jadhav
- Pharmaceutical Chemistry Laboratory, Department of Pharmacy, Birla Institute of Technology and Sciences Pilani, Pilani, RJ, India
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8
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Song G, Lv F, Huang Y, Bai H, Wang S. Conjugated Polymers for Gene Delivery and Photothermal Gene Expression. Chempluschem 2022; 87:e202200073. [DOI: 10.1002/cplu.202200073] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2022] [Revised: 04/26/2022] [Indexed: 11/09/2022]
Affiliation(s)
- Gang Song
- Institute of Chemistry CAS: Institute of Chemistry Chinese Academy of Sciences Organic Solids CHINA
| | - Fengting Lv
- Institute of Chemistry Chinese Academy of Sciences Zhongguancun North First Street 2 CHINA
| | - Yiming Huang
- Institute of Chemistry CAS: Institute of Chemistry Chinese Academy of Sciences Organic Solids CHINA
| | - Haotian Bai
- Institute of Chemistry CAS: Institute of Chemistry Chinese Academy of Sciences Organic Solids CHINA
| | - Shu Wang
- Institute of Chemistry CAS: Institute of Chemistry Chinese Academy of Sciences Organic Solids CHINA
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9
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Fihurka O, Sava V, Sanchez-Ramos J. Dual-function hybrid nanoparticles with gene silencing and anti-inflammatory effects. Nanomedicine (Lond) 2022; 17:577-590. [PMID: 35373577 PMCID: PMC9115733 DOI: 10.2217/nnm-2021-0458] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2021] [Accepted: 03/17/2022] [Indexed: 11/21/2022] Open
Abstract
Background: Nanocarriers loaded with siRNA can be administered intranasally to provide a noninvasive, safe alternative to direct intracerebral or intrathecal infusions. Dual-function nanocarriers can also be designed to deliver several payloads that address different components of the pathological process. Aim: To design and test a hybrid nanocarrier with the capacity to lower Huntington's Disease gene (HTT) expression and prevent or diminish inflammation. Methods: Novel hybrid nanoparticles were fabricated using a chitosan-based matrix core loaded with siRNA and an outer shell consisting of a lipid composition containing cannabidiol. Results: Incubation of hybrid nanoparticles in mesenchymal stem cell cultures obtained from a YAC128 transgenic mouse modeling Huntington's disease resulted in effective lowering of mutant HTT gene expression and reduced levels of expression of the proinflammatory cytokine IL-6. Conclusion: A novel hybrid nanocarrier system with dual actions is effective in lowering HTT gene expression and attenuating inflammatory processes.
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Affiliation(s)
- Oksana Fihurka
- Department of Neurology, University of South Florida, 13220 USF Laurel Drive, Room 4105, Tampa, FL 33612, USA
| | - Vasyl Sava
- Department of Neurology, University of South Florida, 13220 USF Laurel Drive, Room 4105, Tampa, FL 33612, USA
| | - Juan Sanchez-Ramos
- Department of Neurology, University of South Florida, 13220 USF Laurel Drive, Room 4105, Tampa, FL 33612, USA
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Salinas-Luypaert C, Sáez-Cortez F, Quintanilla ME, Herrera-Marschitz M, Rivera-Meza M. Gene knockdown of HCN2 ion channels in the ventral tegmental area reduces ethanol consumption in alcohol preferring rats. THE AMERICAN JOURNAL OF DRUG AND ALCOHOL ABUSE 2022; 48:165-175. [PMID: 35377277 DOI: 10.1080/00952990.2022.2033759] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/15/2021] [Revised: 01/04/2022] [Accepted: 01/15/2022] [Indexed: 06/14/2023]
Abstract
Background: Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) ionic channels are known to play a key role in the control of neuron excitability and have been proposed as a molecular target of ethanol. Previous studies in rats have shown that gene-induced overexpression of the HCN2 channel in the ventral tegmental area (VTA) increases the rewarding effects of ethanol and its intake by the animals.Objective: The aim of this work was to study the effects of VTA HCN2 gene knockdown in the voluntary ethanol consumption of alcohol-preferring UChB rats.Methods: Two lentiviral vectors were generated; LV-siRNA-HCN2, coding for a siRNA that elicited >95% reduction of HCN2 protein levels in vitro, and a control vector coding for a scrambled siRNA sequence. Female UChB naïve rats (n = 14) were microinjected into the VTA with LV-siRNA-HCN2 or the scrambled control vector (n = 11). Four days after, animals were given a daily free access to 10% ethanol and water for 10 days.Results: Rats treated with the LV-siRNA-HCN2 vector showed a ~ 70% reduction (p < .001) in their ethanol preference and ethanol intake compared to control animals. No changes were observed in the total fluid intake of both groups. HCN2 levels in the VTA were measured by Western blot showing a reduction of 40% (p < .05) in the rats injected with LV-siRNA-HCN2, compared to control animals.Conclusion: These results show that knockdown of HCN2 ionic channels in the VTA of UChB rats markedly reduces their voluntary ethanol intake, supporting the idea that HCN2 channels may constitute a therapeutic target for alcohol use disorders.
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Affiliation(s)
- Catalina Salinas-Luypaert
- Department of Pharmacological and Toxicological Chemistry, Faculty of Chemical and Pharmaceutical Sciences Santiago, Chile
| | - Felipe Sáez-Cortez
- Program of Molecular and Clinical Pharmacology, ICBM, Faculty of Medicine, University of Chile, Santiago, Chile
| | - María Elena Quintanilla
- Program of Molecular and Clinical Pharmacology, ICBM, Faculty of Medicine, University of Chile, Santiago, Chile
| | - Mario Herrera-Marschitz
- Program of Molecular and Clinical Pharmacology, ICBM, Faculty of Medicine, University of Chile, Santiago, Chile
| | - Mario Rivera-Meza
- Department of Pharmacological and Toxicological Chemistry, Faculty of Chemical and Pharmaceutical Sciences Santiago, Chile
- Research Center for the Development of Novel Therapeutic Alternatives for Alcohol Use Disorders, Santiago, Chile
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11
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Rahman A, Gupta SD, Rahman MA, Tamanna S. An in-silico approach to design potential siRNAs against the ORF57 of Kaposi's sarcoma-associated herpesvirus. Genomics Inform 2021; 19:e47. [PMID: 35012290 PMCID: PMC8752988 DOI: 10.5808/gi.21057] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2021] [Revised: 11/25/2021] [Accepted: 12/09/2021] [Indexed: 12/21/2022] Open
Abstract
Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few human oncogenic viruses, which causes a variety of malignancies, including Kaposi's sarcoma, multicentric Castleman disease, and primary effusion lymphoma, particularly in human immunodeficiency virus patients. The currently available treatment options cannot always prevent the invasion and dissemination of this virus. In recent times, siRNA-based therapeutics are gaining prominence over conventional medications as siRNA can be designed to target almost any gene of interest. The ORF57 is a crucial regulatory protein for lytic gene expression of KSHV. Disruption of this gene translation will inevitably inhibit the replication of the virus in the host cell. Therefore, the ORF57 of KSHV could be a potential target for designing siRNA-based therapeutics. Considering both sequence preferences and target site accessibility, several online tools (i-SCORE Designer, Sfold web server) had been utilized to predict the siRNA guide strand against the ORF57. Subsequently, off-target filtration (BLAST), conservancy test (fuzznuc), and thermodynamics analysis (RNAcofold, RNAalifold, and RNA Structure web server) were also performed to select the most suitable siRNA sequences. Finally, two siRNAs were identified that passed all of the filtration phases and fulfilled the thermodynamic criteria. We hope that the siRNAs predicted in this study would be helpful for the development of new effective therapeutics against KSHV.
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Affiliation(s)
- Anisur Rahman
- Department of Biotechnology and Genetic Engineering, Faculty of Science, Noakhali Science and Technology University, Noakhali 3814, Bangladesh
| | - Shipan Das Gupta
- Department of Biotechnology and Genetic Engineering, Faculty of Science, Noakhali Science and Technology University, Noakhali 3814, Bangladesh
| | - Md. Anisur Rahman
- Department of Biotechnology and Genetic Engineering, Faculty of Science, Noakhali Science and Technology University, Noakhali 3814, Bangladesh
| | - Saheda Tamanna
- Department of Biotechnology and Genetic Engineering, Faculty of Science, Noakhali Science and Technology University, Noakhali 3814, Bangladesh
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Wang Y, Chai C, Khatabi B, Scheible WR, Udvardi MK, Saha MC, Kang Y, Nelson RS. An Efficient Brome mosaic virus-Based Gene Silencing Protocol for Hexaploid Wheat ( Triticum aestivum L.). FRONTIERS IN PLANT SCIENCE 2021; 12:685187. [PMID: 34220905 PMCID: PMC8253535 DOI: 10.3389/fpls.2021.685187] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/24/2021] [Accepted: 05/07/2021] [Indexed: 05/09/2023]
Abstract
Virus-induced gene silencing (VIGS) is a rapid and powerful method to evaluate gene function, especially for species like hexaploid wheat that have large, redundant genomes and are difficult and time-consuming to transform. The Brome mosaic virus (BMV)-based VIGS vector is widely used in monocotyledonous species but not wheat. Here we report the establishment of a simple and effective VIGS procedure in bread wheat using BMVCP5, the most recently improved BMV silencing vector, and wheat genes PHYTOENE DESATURASE (TaPDS) and PHOSPHATE2 (TaPHO2) as targets. Time-course experiments revealed that smaller inserts (~100 nucleotides, nt) were more stable in BMVCP5 and conferred higher silencing efficiency and longer silencing duration, compared with larger inserts. When using a 100-nt insert and a novel coleoptile inoculation method, BMVCP5 induced extensive silencing of TaPDS transcript and a visible bleaching phenotype in the 2nd to 5th systemically-infected leaves from nine to at least 28 days post inoculation (dpi). For TaPHO2, the ability of BMVCP5 to simultaneously silence all three homoeologs was demonstrated. To investigate the feasibility of BMV VIGS in wheat roots, ectopically expressed enhanced GREEN FLUORESCENT PROTEIN (eGFP) in a transgenic wheat line was targeted for silencing. Silencing of eGFP fluorescence was observed in both the maturation and elongation zones of roots. BMVCP5 mediated significant silencing of eGFP and TaPHO2 mRNA expression in roots at 14 and 21 dpi, and TaPHO2 silencing led to the doubling of inorganic phosphate concentration in the 2nd through 4th systemic leaves. All 54 wheat cultivars screened were susceptible to BMV infection. BMVCP5-mediated TaPDS silencing resulted in the expected bleaching phenotype in all eight cultivars examined, and decreased TaPDS transcript was detected in all three cultivars examined. This BMVCP5 VIGS technology may serve as a rapid and effective functional genomics tool for high-throughput gene function studies in aerial and root tissues and in many wheat cultivars.
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Meng J, Counsell J, Morgan JE. Effects of Mini-Dystrophin on Dystrophin-Deficient, Human Skeletal Muscle-Derived Cells. Int J Mol Sci 2020; 21:E7168. [PMID: 32998454 PMCID: PMC7582244 DOI: 10.3390/ijms21197168] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2020] [Revised: 09/22/2020] [Accepted: 09/24/2020] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND We are developing a novel therapy for Duchenne muscular dystrophy (DMD), involving the transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin. Dystrophin is normally expressed in activated satellite cells and in differentiated muscle fibres. However, in past preclinical validation studies, dystrophin transgenes have generally been driven by constitutive promoters that would be active at every stage of the myogenic differentiation process, including in proliferating muscle stem cells. It is not known whether artificial dystrophin expression would affect the properties of these cells. AIMS Our aims are to determine if mini-dystrophin expression affects the proliferation or myogenic differentiation of DMD skeletal muscle-derived cells. METHODS Skeletal muscle-derived cells from a DMD patient were transduced with lentivirus coding for mini-dystrophins (R3-R13 spectrin-like repeats (ΔR3R13) or hinge2 to spectrin-like repeats R23 (ΔH2R23)) with EGFP (enhanced green fluorescence protein) fused to the C-terminus, driven by a constitutive promoter, spleen focus-forming virus (SFFV). Transduced cells were purified on the basis of GFP expression. Their proliferation and myogenic differentiation were quantified by ethynyl deoxyuridine (EdU) incorporation and fusion index. Furthermore, dystrophin small interfering ribonucleic acids (siRNAs) were transfected to the cells to reverse the effects of the mini-dystrophin. Finally, a phospho-mitogen-activated protein kinase (MAPK) array assay was performed to investigate signalling pathway changes caused by dystrophin expression. RESULTS Cell proliferation was not affected in cells transduced with ΔR3R13, but was significantly increased in cells transduced with ΔH2R23. The fusion index of myotubes derived from both ΔR3R13- and ΔH2R23 -expressing cells was significantly compromised in comparison to myotubes derived from non-transduced cells. Dystrophin siRNA transfection restored the differentiation of ΔH2R23-expressing cells. The Erk1/2- signalling pathway is altered in cells transduced with mini-dystrophin constructs. CONCLUSIONS Ectopic expression of dystrophin in cultured human skeletal muscle-derived cells may affect their proliferation and differentiation capacity. Caution should be taken when considering genetic correction of autologous stem cells to express dystrophin driven by a constitutive promoter.
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MESH Headings
- Cell Differentiation
- Cell Engineering/methods
- Cell Proliferation
- Dystrophin/antagonists & inhibitors
- Dystrophin/genetics
- Dystrophin/metabolism
- Gene Expression Regulation
- Genes, Reporter
- Genetic Vectors/chemistry
- Genetic Vectors/metabolism
- Green Fluorescent Proteins/genetics
- Green Fluorescent Proteins/metabolism
- Humans
- Lentivirus/genetics
- Lentivirus/metabolism
- MAP Kinase Signaling System
- Muscle Fibers, Skeletal/metabolism
- Muscle Fibers, Skeletal/pathology
- Muscle, Skeletal/metabolism
- Muscle, Skeletal/pathology
- Muscular Dystrophy, Duchenne/genetics
- Muscular Dystrophy, Duchenne/metabolism
- Muscular Dystrophy, Duchenne/pathology
- Plasmids/chemistry
- Plasmids/metabolism
- Primary Cell Culture
- RNA, Small Interfering/genetics
- RNA, Small Interfering/metabolism
- Spectrin/genetics
- Spectrin/metabolism
- Transduction, Genetic
- Transgenes
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Affiliation(s)
- Jinhong Meng
- Dubowitz Neuromuscular Centre, Developmental Neuroscience Research and Teaching Department, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK; (J.M.); (J.C.)
- NIHR Great Ormond Street Hospital Biomedical Research Centre, UCL Great Ormond Street Institute of Child Health & Great Ormond Street Hospital for Children NHS Foundation Trust, London WC1N 1EH, UK
| | - John Counsell
- Dubowitz Neuromuscular Centre, Developmental Neuroscience Research and Teaching Department, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK; (J.M.); (J.C.)
- NIHR Great Ormond Street Hospital Biomedical Research Centre, UCL Great Ormond Street Institute of Child Health & Great Ormond Street Hospital for Children NHS Foundation Trust, London WC1N 1EH, UK
| | - Jennifer E. Morgan
- Dubowitz Neuromuscular Centre, Developmental Neuroscience Research and Teaching Department, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK; (J.M.); (J.C.)
- NIHR Great Ormond Street Hospital Biomedical Research Centre, UCL Great Ormond Street Institute of Child Health & Great Ormond Street Hospital for Children NHS Foundation Trust, London WC1N 1EH, UK
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14
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Shmushkovich T, Monopoli KR, Homsy D, Leyfer D, Betancur-Boissel M, Khvorova A, Wolfson AD. Functional features defining the efficacy of cholesterol-conjugated, self-deliverable, chemically modified siRNAs. Nucleic Acids Res 2019; 46:10905-10916. [PMID: 30169779 PMCID: PMC6237813 DOI: 10.1093/nar/gky745] [Citation(s) in RCA: 52] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2018] [Accepted: 08/24/2018] [Indexed: 12/14/2022] Open
Abstract
Progress in oligonucleotide chemistry has produced a shift in the nature of siRNA used, from formulated, minimally modified siRNAs, to unformulated, heavily modified siRNA conjugates. The introduction of extensive chemical modifications is essential for conjugate-mediated delivery. Modifications have a significant impact on siRNA efficacy through interference with recognition and processing by RNAi enzymatic machinery, severely restricting the sequence space available for siRNA design. Many algorithms available publicly can successfully predict the activity of non-modified siRNAs, but the efficiency of the algorithms for designing heavily modified siRNAs has never been systematically evaluated experimentally. Here we screened 356 cholesterol-conjugated siRNAs with extensive modifications and developed a linear regression-based algorithm that effectively predicts siRNA activity using two independent datasets. We further demonstrate that predictive determinants for modified and non-modified siRNAs differ substantially. The algorithm developed from the non-modified siRNAs dataset has no predictive power for modified siRNAs and vice versa. In the context of heavily modified siRNAs, the introduction of chemical asymmetry fully eliminates the requirement for thermodynamic bias, the major determinant for non-modified siRNA efficacy. Finally, we demonstrate that in addition to the sequence of the target site, the accessibility of the neighboring 3′ region significantly contributes to siRNA efficacy.
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Affiliation(s)
| | | | - Diana Homsy
- Advirna, 60 Prescott Street, Worcester, MA 01605, USA
| | - Dmitriy Leyfer
- Advirna, 60 Prescott Street, Worcester, MA 01605, USA.,Boston University, 44 Cummington Mall, Boston, MA 02215, USA
| | | | - Anastasia Khvorova
- University of Massachusetts Medical School, 368 Plantation Street. Worcester, MA 01655, USA
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15
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Guru Vishnu P, Bhattacharya TK, Bhushan B, Kumar P, Chatterjee RN, Paswan C, Dushyanth K, Divya D, Prasad AR. In silico prediction of short hairpin RNA and in vitro silencing of activin receptor type IIB in chicken embryo fibroblasts by RNA interference. Mol Biol Rep 2019; 46:2947-2959. [PMID: 30879273 DOI: 10.1007/s11033-019-04756-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2019] [Accepted: 03/08/2019] [Indexed: 12/26/2022]
Abstract
Gene silencing by RNA interference is extensively used reverse genetic approach to analyse the implications of any gene in mammalian systems. The silencing of the Activin type IIB receptor belonging to transforming growth factor beta superfamily has demonstrated increase in muscle growth in many species. We designed five short hairpin RNA constructs targeting coding region of chicken ACTRIIB. All the shRNAs were transfected into chicken embryo fibroblast cells and evaluated their silencing efficiency by real time PCR and western blotting. Initially the computational analysis of target region and shRNA constructs was undertaken to predict sequence based features (secondary structures, GC% and H-b index) and thermodynamic features (ΔGoverall, ΔGduplex, ΔGbreak-target, ΔGintra-oligomer, ΔGinter-oligomer and ΔΔGends). We determined that all these predicted features were associated with shRNA efficacy. The invitro analysis of shRNA constructs exhibited significant (P < 0.05) reduction in the levels of ACTRIIB at mRNA and protein level. The knock down efficiency of shRNAs varied significantly (P < 0.001) from 83% (shRNA 1) to 43% (shRNA 5). All the shRNAs up regulated the myogenic pathway associated genes (MyoD and MyoG) significantly (P < 0.05). There was significant (P < 0.05) up-regulation of IFNA, IFNB and MHCII transcripts. The ACTRIIB expression was inversely associated with the expression of myogenic pathway and immune response genes. The anti ACTRIIB shRNA construct 1 and 3 exhibited maximum knock down efficiency with minimal interferon response, and can be used for generating ACTRIIB knockdown chicken with higher muscle mass.
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Affiliation(s)
- P Guru Vishnu
- Sri Venkateswara Veterinary University, Tirupathi, A.P., India.
| | | | - Bharat Bhushan
- Division of Animal Genetics & Breeding, Indian Veterinary Research Institute, Izatnagar, U.P., India
| | - Pushpendra Kumar
- Division of Animal Genetics & Breeding, Indian Veterinary Research Institute, Izatnagar, U.P., India
| | | | | | - K Dushyanth
- ICAR-Directorate of Poultry Research, Hyderabad, India
| | - D Divya
- ICAR-Directorate of Poultry Research, Hyderabad, India
| | - A Rajendra Prasad
- Division of Animal Genetics & Breeding, Indian Veterinary Research Institute, Izatnagar, U.P., India
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16
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Affiliation(s)
- Arthur A Levin
- From Research and Development, Avidity Biosciences, La Jolla, CA
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17
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To accelerate the Zika beat: Candidate design for RNA interference-based therapy. Virus Res 2018; 255:133-140. [DOI: 10.1016/j.virusres.2018.07.010] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2018] [Revised: 06/11/2018] [Accepted: 07/17/2018] [Indexed: 12/12/2022]
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18
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Braga ACS, Carneiro BM, Batista MN, Akinaga MM, Rahal P. Inhibition of hepatitis C virus using siRNA targeted to the virus and Hsp90. Cell Stress Chaperones 2017; 22:113-122. [PMID: 27858224 PMCID: PMC5225065 DOI: 10.1007/s12192-016-0747-8] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2016] [Revised: 10/24/2016] [Accepted: 11/08/2016] [Indexed: 01/19/2023] Open
Abstract
Hepatitis C (HCV) is a viral disease affecting millions of people worldwide, and persistent HCV infection can lead to progressive liver disease with the development of liver cirrhosis and hepatocellular carcinoma. During treatment for hepatitis C, the occurrence of viral resistance is common. To reduce the occurrence of resistance, new viral treatments should target both viral and cellular factors. Many interactions occur between viral and host proteins during the HCV replication cycle and might be used for the development of new therapies against hepatitis C. Heat shock protein 90 (Hsp90) plays a role in the folding of cellular and viral proteins and also interacts with HCV proteins. In the present study, we knocked down the expression of the Hsp90 gene and inhibited viral replication using siRNA molecules. Reducing the expression of Hsp90 successfully decreased HCV replication. All siRNA molecules specific to the viral genome showed the efficient inhibition of viral replication, particularly siRNA targeted to the 5'UTR region. The combination of siRNAs targeting the viral genome and Hsp90 mRNA also successfully reduced HCV replication and reduced the occurrence of viral resistance. Moreover, these results suggest that an approach based on the combination of cellular and viral siRNAs can be used as an effective alternative for hepatitis C viral suppression.
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Affiliation(s)
- Ana Claudia Silva Braga
- Institute of Biosciences, Letters and Exact Sciences, UNESP, Rua Cristóvão Colombo, 2265, São José do Rio Preto, SP, CEP: 15054-000, Brazil
| | - Bruno Moreira Carneiro
- Institute of Biosciences, Letters and Exact Sciences, UNESP, Rua Cristóvão Colombo, 2265, São José do Rio Preto, SP, CEP: 15054-000, Brazil
- Institute of Exact and Natural Sciences, Mato Grosso Federal University, Rondonópolis, Brazil
| | - Mariana Nogueira Batista
- Institute of Biosciences, Letters and Exact Sciences, UNESP, Rua Cristóvão Colombo, 2265, São José do Rio Preto, SP, CEP: 15054-000, Brazil
| | - Mônica Mayumi Akinaga
- Institute of Biosciences, Letters and Exact Sciences, UNESP, Rua Cristóvão Colombo, 2265, São José do Rio Preto, SP, CEP: 15054-000, Brazil
| | - Paula Rahal
- Institute of Biosciences, Letters and Exact Sciences, UNESP, Rua Cristóvão Colombo, 2265, São José do Rio Preto, SP, CEP: 15054-000, Brazil.
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19
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Fakhr E, Zare F, Teimoori-Toolabi L. Precise and efficient siRNA design: a key point in competent gene silencing. Cancer Gene Ther 2016; 23:73-82. [DOI: 10.1038/cgt.2016.4] [Citation(s) in RCA: 130] [Impact Index Per Article: 14.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2015] [Revised: 02/01/2016] [Accepted: 02/01/2016] [Indexed: 12/14/2022]
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20
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Ragusa M, Barbagallo C, Statello L, Condorelli AG, Battaglia R, Tamburello L, Barbagallo D, Di Pietro C, Purrello M. Non-coding landscapes of colorectal cancer. World J Gastroenterol 2015; 21:11709-11739. [PMID: 26556998 PMCID: PMC4631972 DOI: 10.3748/wjg.v21.i41.11709] [Citation(s) in RCA: 67] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Revised: 07/28/2015] [Accepted: 09/30/2015] [Indexed: 02/06/2023] Open
Abstract
For two decades Vogelstein’s model has been the paradigm for describing the sequence of molecular changes within protein-coding genes that would lead to overt colorectal cancer (CRC). This model is now too simplistic in the light of recent studies, which have shown that our genome is pervasively transcribed in RNAs other than mRNAs, denominated non-coding RNAs (ncRNAs). The discovery that mutations in genes encoding these RNAs [i.e., microRNAs (miRNAs), long non-coding RNAs, and circular RNAs] are causally involved in cancer phenotypes has profoundly modified our vision of tumour molecular genetics and pathobiology. By exploiting a wide range of different mechanisms, ncRNAs control fundamental cellular processes, such as proliferation, differentiation, migration, angiogenesis and apoptosis: these data have also confirmed their role as oncogenes or tumor suppressors in cancer development and progression. The existence of a sophisticated RNA-based regulatory system, which dictates the correct functioning of protein-coding networks, has relevant biological and biomedical consequences. Different miRNAs involved in neoplastic and degenerative diseases exhibit potential predictive and prognostic properties. Furthermore, the key roles of ncRNAs make them very attractive targets for innovative therapeutic approaches. Several recent reports have shown that ncRNAs can be secreted by cells into the extracellular environment (i.e., blood and other body fluids): this suggests the existence of extracellular signalling mechanisms, which may be exploited by cells in physiology and pathology. In this review, we will summarize the most relevant issues on the involvement of cellular and extracellular ncRNAs in disease. We will then specifically describe their involvement in CRC pathobiology and their translational applications to CRC diagnosis, prognosis and therapy.
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Abstract
Post-transcriptional gene silencing is a widely used method to suppress gene expression. Unfortunately only a portion of siRNAs do successfully reduce gene expression. Target mRNA secondary structures and siRNA-mRNA thermodynamic features are believed to contribute to the silencing activity. However, there is still an open discussion as to what determines siRNA efficacy. In this retrospective study, we analysed the target accessibility comparing very high (VH) compared with low (L) efficacy siRNA sequences obtained from the siRecords Database. We determined the contribution of mRNA target local secondary structures on silencing efficacy. Both the univariable and the multivariable logistic regression evidenced no relationship between siRNA efficacy and mRNA target secondary structures. Moreover, none of the thermodynamic and sequence-base parameters taken into consideration (H-b index, ΔG°overall, ΔG°duplex, ΔG°break-target and GC%) was associated with siRNA efficacy. We found that features believed to be predictive of silencing efficacy are not confirmed to be so when externally evaluated in a large heterogeneous sample. Although it was proposed that silencing efficacy could be influenced by local target accessibility we show that this could be not generalizable because of the diversity of experimental setting that may not be representative of biological systems especially in view of the many local protein factors, usually not taken into consideration, which could hamper the silencing process. We analysed several siRNA-mRNA target features involved in silencing efficacy. We found out that features believed to be predictive of silencing efficacy are not such when transferred to a larger dataset of experiments and different experimental settings.
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22
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Fiszer A, Krzyzosiak WJ. Oligonucleotide-based strategies to combat polyglutamine diseases. Nucleic Acids Res 2014; 42:6787-810. [PMID: 24848018 PMCID: PMC4066792 DOI: 10.1093/nar/gku385] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Considerable advances have been recently made in understanding the molecular aspects of pathogenesis and in developing therapeutic approaches for polyglutamine (polyQ) diseases. Studies on pathogenic mechanisms have extended our knowledge of mutant protein toxicity, confirmed the toxicity of mutant transcript and identified other toxic RNA and protein entities. One very promising therapeutic strategy is targeting the causative gene expression with oligonucleotide (ON) based tools. This straightforward approach aimed at halting the early steps in the cascade of pathogenic events has been widely tested for Huntington's disease and spinocerebellar ataxia type 3. In this review, we gather information on the use of antisense oligonucleotides and RNA interference triggers for the experimental treatment of polyQ diseases in cellular and animal models. We present studies testing non-allele-selective and allele-selective gene silencing strategies. The latter include targeting SNP variants associated with mutations or targeting the pathologically expanded CAG repeat directly. We compare gene silencing effectors of various types in a number of aspects, including their design, efficiency in cell culture experiments and pre-clinical testing. We discuss advantages, current limitations and perspectives of various ON-based strategies used to treat polyQ diseases.
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Affiliation(s)
- Agnieszka Fiszer
- Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland
| | - Wlodzimierz J Krzyzosiak
- Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland
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23
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Watanabe T, Hatakeyama H, Matsuda-Yasui C, Sato Y, Sudoh M, Takagi A, Hirata Y, Ohtsuki T, Arai M, Inoue K, Harashima H, Kohara M. In vivo therapeutic potential of Dicer-hunting siRNAs targeting infectious hepatitis C virus. Sci Rep 2014; 4:4750. [PMID: 24756133 PMCID: PMC3996463 DOI: 10.1038/srep04750] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2014] [Accepted: 04/04/2014] [Indexed: 01/07/2023] Open
Abstract
The development of RNA interference (RNAi)-based therapy faces two major obstacles: selecting small interfering RNA (siRNA) sequences with strong activity, and identifying a carrier that allows efficient delivery to target organs. Additionally, conservative region at nucleotide level must be targeted for RNAi in applying to virus because hepatitis C virus (HCV) could escape from therapeutic pressure with genome mutations. In vitro preparation of Dicer-generated siRNAs targeting a conserved, highly ordered HCV 5' untranslated region are capable of inducing strong RNAi activity. By dissecting the 5'-end of an RNAi-mediated cleavage site in the HCV genome, we identified potent siRNA sequences, which we designate as Dicer-hunting siRNAs (dh-siRNAs). Furthermore, formulation of the dh-siRNAs in an optimized multifunctional envelope-type nano device inhibited ongoing infectious HCV replication in human hepatocytes in vivo. Our efforts using both identification of optimal siRNA sequences and delivery to human hepatocytes suggest therapeutic potential of siRNA for a virus.
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Affiliation(s)
- Tsunamasa Watanabe
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan
- Division of Gastroenterology, Showa University Fujigaoka Hospital, Yokohama, Japan
- Present address, Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Sciences, Kawasumi, Mizuho, Nagoya 467-8601, Japan
- These authors contributed equally to this work
| | - Hiroto Hatakeyama
- Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido 060-0812, Japan
- These authors contributed equally to this work
| | - Chiho Matsuda-Yasui
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan
- These authors contributed equally to this work
| | - Yusuke Sato
- Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido 060-0812, Japan
- These authors contributed equally to this work
| | - Masayuki Sudoh
- Kamakura Research Laboratories, Chugai Pharmaceutical Co., Ltd., Kamakura, Kanagawa 247-8530, Japan
| | - Asako Takagi
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan
| | - Yuichi Hirata
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan
- Division of Gastroenterology, Showa University Fujigaoka Hospital, Yokohama, Japan
| | - Takahiro Ohtsuki
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan
| | - Masaaki Arai
- Advanced Medical Research Laboratory, Mitsubishi Tanabe Pharma Corporation, 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan
| | - Kazuaki Inoue
- Division of Gastroenterology, Showa University Fujigaoka Hospital, Yokohama, Japan
| | - Hideyoshi Harashima
- Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido 060-0812, Japan
| | - Michinori Kohara
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan
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Malefyt AP, Wu M, Vocelle DB, Kappes SJ, Lindeman SD, Chan C, Walton SP. Improved asymmetry prediction for short interfering RNAs. FEBS J 2014; 281:320-30. [PMID: 24393396 DOI: 10.1111/febs.12599] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2013] [Revised: 08/28/2013] [Accepted: 09/26/2013] [Indexed: 01/10/2023]
Abstract
In the development of RNA interference therapeutics, merely selecting short interfering RNA (siRNA) sequences that are complementary to the mRNA target does not guarantee target silencing. Current algorithms for selecting siRNAs rely on many parameters, one of which is asymmetry, often predicted through calculation of the relative thermodynamic stabilities of the two ends of the siRNA. However, we have previously shown that highly active siRNA sequences are likely to have particular nucleotides at each 5'-end, independently of their thermodynamic asymmetry. Here, we describe an algorithm for predicting highly active siRNA sequences based only on these two asymmetry parameters. The algorithm uses end-sequence nucleotide preferences and predicted thermodynamic stabilities, each weighted on the basis of training data from the literature, to rank the probability that an siRNA sequence will have high or low activity. The algorithm successfully predicts weakly and highly active sequences for enhanced green fluorescent protein and protein kinase R. Use of these two parameters in combination improves the prediction of siRNA activity over current approaches for predicting asymmetry. Going forward, we anticipate that this approach to siRNA asymmetry prediction will be incorporated into the next generation of siRNA selection algorithms.
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Affiliation(s)
- Amanda P Malefyt
- Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI, USA
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Angart P, Vocelle D, Chan C, Walton SP. Design of siRNA Therapeutics from the Molecular Scale. Pharmaceuticals (Basel) 2013; 6:440-68. [PMID: 23976875 PMCID: PMC3749788 DOI: 10.3390/ph6040440] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
While protein-based therapeutics is well-established in the market, development of nucleic acid therapeutics has lagged. Short interfering RNAs (siRNAs) represent an exciting new direction for the pharmaceutical industry. These small, chemically synthesized RNAs can knock down the expression of target genes through the use of a native eukaryotic pathway called RNA interference (RNAi). Though siRNAs are routinely used in research studies of eukaryotic biological processes, transitioning the technology to the clinic has proven challenging. Early efforts to design an siRNA therapeutic have demonstrated the difficulties in generating a highly-active siRNA with good specificity and a delivery vehicle that can protect the siRNA as it is transported to a specific tissue. In this review article, we discuss design considerations for siRNA therapeutics, identifying criteria for choosing therapeutic targets, producing highly-active siRNA sequences, and designing an optimized delivery vehicle. Taken together, these design considerations provide logical guidelines for generating novel siRNA therapeutics.
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Affiliation(s)
- Phillip Angart
- Department of Chemical Engineering and Materials Science, Michigan State University, 428 S. Shaw Lane, Room 2527, East Lansing, MI 48824, USA; (P.A.); (D.V.); (C.C.)
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26
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Li CH, Chen Y. Targeting long non-coding RNAs in cancers: Progress and prospects. Int J Biochem Cell Biol 2013; 45:1895-910. [DOI: 10.1016/j.biocel.2013.05.030] [Citation(s) in RCA: 344] [Impact Index Per Article: 28.7] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2013] [Revised: 05/21/2013] [Accepted: 05/23/2013] [Indexed: 02/07/2023]
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Sciabola S, Cao Q, Orozco M, Faustino I, Stanton RV. Improved nucleic acid descriptors for siRNA efficacy prediction. Nucleic Acids Res 2012; 41:1383-94. [PMID: 23241392 PMCID: PMC3561943 DOI: 10.1093/nar/gks1191] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Although considerable progress has been made recently in understanding how gene silencing is mediated by the RNAi pathway, the rational design of effective sequences is still a challenging task. In this article, we demonstrate that including three-dimensional descriptors improved the discrimination between active and inactive small interfering RNAs (siRNAs) in a statistical model. Five descriptor types were used: (i) nucleotide position along the siRNA sequence, (ii) nucleotide composition in terms of presence/absence of specific combinations of di- and trinucleotides, (iii) nucleotide interactions by means of a modified auto- and cross-covariance function, (iv) nucleotide thermodynamic stability derived by the nearest neighbor model representation and (v) nucleic acid structure flexibility. The duplex flexibility descriptors are derived from extended molecular dynamics simulations, which are able to describe the sequence-dependent elastic properties of RNA duplexes, even for non-standard oligonucleotides. The matrix of descriptors was analysed using three statistical packages in R (partial least squares, random forest, and support vector machine), and the most predictive model was implemented in a modeling tool we have made publicly available through SourceForge. Our implementation of new RNA descriptors coupled with appropriate statistical algorithms resulted in improved model performance for the selection of siRNA candidates when compared with publicly available siRNA prediction tools and previously published test sets. Additional validation studies based on in-house RNA interference projects confirmed the robustness of the scoring procedure in prospective studies.
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Affiliation(s)
- Simone Sciabola
- Pfizer Oligonucleotide Therapeutic Unit, 620 Memorial Drive, Cambridge, Massachusetts 02139, USA.
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Marín RM, Voellmy F, von Erlach T, Vaníček J. Analysis of the accessibility of CLIP bound sites reveals that nucleation of the miRNA:mRNA pairing occurs preferentially at the 3'-end of the seed match. RNA (NEW YORK, N.Y.) 2012; 18:1760-1770. [PMID: 22915600 PMCID: PMC3446701 DOI: 10.1261/rna.033282.112] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/15/2012] [Accepted: 08/01/2012] [Indexed: 06/01/2023]
Abstract
To find out whether the AGO-miRNA complex is more sensitive to the accessibility of a particular region inside the seed match, we analyze in detail the accessibility of a wide set of miRNA binding sites validated by PAR-CLIP and HITS-CLIP experiments. Our analysis reveals that nucleotides at the 3'-end of bound seed matches are significantly more accessible than nucleotides at the 5'-end as well as nucleotides at any positions in the unbound seed matches. We show that the accessibility of a single nucleotide at the 3'-end is more effective than the accessibility of several nucleotides at the 5'-end in discriminating between functional and nonfunctional binding sites. Analysis of mRNA and protein fold changes induced by miRNA overexpression demonstrates that genes with accessible nucleation regions at the 3'-end are down-regulated more strongly than genes whose accessible nucleation regions are located elsewhere within the seed match. We also observed an increase in the precision of the miRNA target prediction algorithm PACMIT when accessibility toward the 3'-end of the seed match was required. The pronounced sensitivity of the AGO-miRNA complex to the accessibility of the 3'-end of the seed match suggests that, in most cases, nucleation occurs in this region. We show that this conclusion is consistent with previous experimental studies.
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Affiliation(s)
- Ray M. Marín
- Laboratory of Theoretical Physical Chemistry, Institut des Sciences et Ingénierie Chimiques, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
| | - Franziska Voellmy
- Laboratory of Theoretical Physical Chemistry, Institut des Sciences et Ingénierie Chimiques, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
| | - Thibaud von Erlach
- Laboratory of Theoretical Physical Chemistry, Institut des Sciences et Ingénierie Chimiques, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
| | - Jiří Vaníček
- Laboratory of Theoretical Physical Chemistry, Institut des Sciences et Ingénierie Chimiques, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
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Abstract
SUMMARYAlmost a decade has passed since the first report of RNA interference (RNAi) in a parasitic helminth. Whilst much progress has been made with RNAi informing gene function studies in disparate nematode and flatworm parasites, substantial and seemingly prohibitive difficulties have been encountered in some species, hindering progress. An appraisal of current practices, trends and ideals of RNAi experimental design in parasitic helminths is both timely and necessary for a number of reasons: firstly, the increasing availability of parasitic helminth genome/transcriptome resources means there is a growing need for gene function tools such as RNAi; secondly, fundamental differences and unique challenges exist for parasite species which do not apply to model organisms; thirdly, the inherent variation in experimental design, and reported difficulties with reproducibility undermine confidence. Ideally, RNAi studies of gene function should adopt standardised experimental design to aid reproducibility, interpretation and comparative analyses. Although the huge variations in parasite biology and experimental endpoints make RNAi experimental design standardization difficult or impractical, we must strive to validate RNAi experimentation in helminth parasites. To aid this process we identify multiple approaches to RNAi experimental validation and highlight those which we deem to be critical for gene function studies in helminth parasites.
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Abstract
RNA interference (RNAi) has been extensively employed for in vivo research since its use was first demonstrated in mammalian cells 10 years ago. Design rules have improved, and it is now routinely possible to obtain reagents that suppress expression of any gene desired. At the same time, increased understanding of the molecular basis of unwanted side effects has led to the development of chemical modification strategies that mitigate these concerns. Delivery remains the single greatest hurdle to widespread adoption of in vivo RNAi methods. However, exciting advances have been made and new delivery systems under development may help to overcome these barriers. This review discusses advances in RNAi biochemistry and biology that impact in vivo use and provides an overview of select publications that demonstrate interesting applications of these principles. Emphasis is placed on work with synthetic, small interfering RNAs (siRNAs) published since the first installment of this review which appeared in 2006.
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Shah PS, Schaffer DV. Antiviral RNAi: translating science towards therapeutic success. Pharm Res 2011; 28:2966-82. [PMID: 21826573 PMCID: PMC5012899 DOI: 10.1007/s11095-011-0549-8] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2011] [Accepted: 07/25/2011] [Indexed: 01/07/2023]
Abstract
Viruses continuously evolve to contend with an ever-changing environment that involves transmission between hosts and sometimes species, immune responses, and in some cases therapeutic interventions. Given the high mutation rate of viruses relative to the timescales of host evolution and drug development, novel drug classes that are readily screened and translated to the clinic are needed. RNA interference (RNAi)-a natural mechanism for specific degradation of target RNAs that is conserved from plants to invertebrates and vertebrates-can potentially be harnessed to yield therapies with extensive specificity, ease of design, and broad application. In this review, we discuss basic mechanisms of action and therapeutic applications of RNAi, including design considerations and areas for future development in the field.
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Affiliation(s)
- Priya S. Shah
- Department of Chemical and Biolmolecular Engineering, University of California, Berkeley, California 94720 USA
| | - David V. Schaffer
- Department of Chemical and Biolmolecular Engineering, University of California, Berkeley, California 94720 USA
- Department of Bioengineering, University of California, Berkeley, California 94720 USA
- Helen Wills Neuroscience Institute, University of California, Berkeley, California 94720 USA
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Abstract
The design of small interfering RNA (siRNA) is a multi factorial problem that has gained the attention of many researchers in the area of therapeutic and functional genomics. MysiRNA score was previously introduced that improves the correlation of siRNA activity prediction considering state of the art algorithms. In this paper, a new program, MysiRNA-Designer, is described which integrates several factors in an automated work-flow considering mRNA transcripts variations, siRNA and mRNA target accessibility, and both near-perfect and partial off-target matches. It also features the MysiRNA score, a highly ranked correlated siRNA efficacy prediction score for ranking the designed siRNAs, in addition to top scoring models Biopredsi, DISR, Thermocomposition21 and i-Score, and integrates them in a unique siRNA score-filtration technique. This multi-score filtration layer filters siRNA that passes the 90% thresholds calculated from experimental dataset features. MysiRNA-Designer takes an accession, finds conserved regions among its transcript space, finds accessible regions within the mRNA, designs all possible siRNAs for these regions, filters them based on multi-scores thresholds, and then performs SNP and off-target filtration. These strict selection criteria were tested against human genes in which at least one active siRNA was designed from 95.7% of total genes. In addition, when tested against an experimental dataset, MysiRNA-Designer was found capable of rejecting 98% of the false positive siRNAs, showing superiority over three state of the art siRNA design programs. MysiRNA is a freely accessible (Microsoft Windows based) desktop application that can be used to design siRNA with a high accuracy and specificity. We believe that MysiRNA-Designer has the potential to play an important role in this area.
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ElHefnawi M, Hassan N, Kamar M, Siam R, Remoli AL, El-Azab I, AlAidy O, Marsili G, Sgarbanti M. The design of optimal therapeutic small interfering RNA molecules targeting diverse strains of influenza A virus. Bioinformatics 2011; 27:3364-70. [DOI: 10.1093/bioinformatics/btr555] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
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Kiryu H, Terai G, Imamura O, Yoneyama H, Suzuki K, Asai K. A detailed investigation of accessibilities around target sites of siRNAs and miRNAs. ACTA ACUST UNITED AC 2011; 27:1788-97. [PMID: 21531769 DOI: 10.1093/bioinformatics/btr276] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
MOTIVATION The importance of RNA sequence analysis has been increasing since the discovery of various types of non-coding RNAs transcribed in animal cells. Conventional RNA sequence analyses have mainly focused on structured regions, which are stabilized by the stacking energies acting on adjacent base pairs. On the other hand, recent findings regarding the mechanisms of small interfering RNAs (siRNAs) and transcription regulation by microRNAs (miRNAs) indicate the importance of analyzing accessible regions where no base pairs exist. So far, relatively few studies have investigated the nature of such regions. RESULTS We have conducted a detailed investigation of accessibilities around the target sites of siRNAs and miRNAs. We have exhaustively calculated the correlations between the accessibilities around the target sites and the repression levels of the corresponding mRNAs. We have computed the accessibilities with an originally developed software package, called 'Raccess', which computes the accessibility of all the segments of a fixed length for a given RNA sequence when the maximal distance between base pairs is limited to a fixed size W. We show that the computed accessibilities are relatively insensitive to the choice of the maximal span W. We have found that the efficacy of siRNAs depends strongly on the accessibility of the very 3'-end of their binding sites, which might reflect a target site recognition mechanism in the RNA-induced silencing complex. We also show that the efficacy of miRNAs has a similar dependence on the accessibilities, but some miRNAs also show positive correlations between the efficacy and the accessibilities in broad regions downstream of their putative binding sites, which might imply that the downstream regions of the target sites are bound by other proteins that allow the miRNAs to implement their functions. We have also investigated the off-target effects of an siRNA as a potential RNAi therapeutic. We show that the off-target effects of the siRNA have similar correlations to the miRNA repression, indicating that they are caused by the same mechanism. AVAILABILITY The C++ source code of the Raccess software is available at http://www.ncrna.org/software/Raccess/ The microarray data on the measurements of the siRNA off-target effects are also available at the same site. CONTACT kiryu-h@k.u-tokyo.ac.jp
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Affiliation(s)
- Hisanori Kiryu
- Department of Computational Biology, Faculty of Frontier Science, The University of Tokyo, Chiba 277-8561, Japan.
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Abstract
The discovery of RNA interference (RNAi) generated considerable interest in developing short interfering RNAs (siRNAs) for understanding basic biology and as the active agents in a new variety of therapeutics. Early studies showed that selecting an active siRNA was not as straightforward as simply picking a sequence on the target mRNA and synthesizing the siRNA complementary to that sequence. As interest in applying RNAi has increased, the methods for identifying active siRNA sequences have evolved from focusing on the simplicity of synthesis and purification, to identifying preferred target sequences and secondary structures, to predicting the thermodynamic stability of the siRNA. As more specific details of the RNAi mechanism have been defined, these have been incorporated into more complex siRNA selection algorithms, increasing the reliability of selecting active siRNAs against a single target. Ultimately, design of the best siRNA therapeutics will require design of the siRNA itself, in addition to design of the vehicle and other components necessary for it to function in vivo. In this minireview, we summarize the evolution of siRNA selection techniques with a particular focus on one issue of current importance to the field, how best to identify those siRNA sequences likely to have high activity. Approaches to designing active siRNAs through chemical and structural modifications will also be highlighted. As the understanding of how to control the activity and specificity of siRNAs improves, the potential utility of siRNAs as human therapeutics will concomitantly grow.
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Affiliation(s)
- S Patrick Walton
- Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824-1226, USA.
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Pacak A, Geisler K, Jørgensen B, Barciszewska-Pacak M, Nilsson L, Nielsen TH, Johansen E, Grønlund M, Jakobsen I, Albrechtsen M. Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat. PLANT METHODS 2010; 6:26. [PMID: 21118486 PMCID: PMC3006357 DOI: 10.1186/1746-4811-6-26] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/13/2010] [Accepted: 11/30/2010] [Indexed: 05/18/2023]
Abstract
BACKGROUND Gene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species. RESULTS Here we demonstrate the successful BSMV-mediated virus induced gene silencing (VIGS) of three different genes in barley roots, i.e. the barley homologues of the IPS1, PHR1, and PHO2 genes known to participate in Pi uptake and reallocation in Arabidopsis. Attempts to silence two other genes, the Pi transporter gene HvPht1;1 and the endo-β-1,4-glucanase gene HvCel1, in barley roots were unsuccessful, probably due to instability of the plant gene inserts in the viral vector. In B. distachyon leaves, significant silencing of the PHYTOENE DESATURASE (BdPDS) gene was obtained as shown by photobleaching as well as quantitative RT-PCR analysis. On the other hand, only very limited silencing of the oat AsPDS gene was observed in both hexaploid (A. sativa) and diploid (A. strigosa) oat. Finally, two modifications of the BSMV vector are presented, allowing ligation-free cloning of DNA fragments into the BSMV-γ component. CONCLUSIONS Our results show that BSMV can be used as a vector for gene silencing in barley roots and in B. distachyon leaves and possibly roots, opening up possibilities for using VIGS to study cereal root biology and to exploit the wealth of genome information in the new cereal model plant B. distachyon. On the other hand, the silencing induced by BSMV in oat seemed too weak to be of practical use. The new BSMV vectors modified for ligation-free cloning will allow rapid insertion of plant gene fragments for future experiments.
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Affiliation(s)
- Andrzej Pacak
- Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
- Current Address: Department of Gene Expression, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland
| | - Katrin Geisler
- Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
- Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Denmark
| | - Bodil Jørgensen
- Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
- Current Address: Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
| | - Maria Barciszewska-Pacak
- Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Denmark
- Current Address: Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, S901-83 Umeå, Sweden
| | - Lena Nilsson
- Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Denmark
- Current Address: Section for Sustainable Biotechnology, Department of Biotechnology, Chemistry and Environmental Engineering, Copenhagen Institute of Technology, Aalborg University, Ballerup, Denmark
| | - Tom Hamborg Nielsen
- Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Denmark
| | - Elisabeth Johansen
- Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
| | - Mette Grønlund
- Biosystems Division, Risø National Laboratory for Sustainable Energy, Technical University of Denmark, PO Box 49, DK-4000 Roskilde, Denmark
| | - Iver Jakobsen
- Biosystems Division, Risø National Laboratory for Sustainable Energy, Technical University of Denmark, PO Box 49, DK-4000 Roskilde, Denmark
| | - Merete Albrechtsen
- Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
- Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Denmark
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Kim J, Shin JS. Probing the transition state for nucleic acid hybridization using phi-value analysis. Biochemistry 2010; 49:3420-6. [PMID: 20210364 DOI: 10.1021/bi902047x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Genetic regulation by noncoding RNA elements such as microRNA and small interfering RNA (siRNA) involves hybridization of a short single-stranded RNA with a complementary segment in a target mRNA. The physical basis of the hybridization process between the structured nucleic acids is not well understood primarily because of the lack of information about the transition-state structure. Here we use transition-state theory, inspired by phi-value analysis in protein folding studies, to provide quantitative analysis of the relationship between changes in the secondary structure stability and the activation free energy. Time course monitoring of the hybridization reaction was performed under pseudo-steady-state conditions using a single fluorophore. The phi-value analysis indicates that the native secondary structure remains intact in the transition state. The nativelike transition state was confirmed via examination of the salt dependence of the hybridization kinetics, indicating that the number of sodium ions associated with the transition state was not substantially affected by changes in the native secondary structure. These results propose that hybridization between structured nucleic acids undergoes a transition state leading to formation of a nucleation complex and then is followed by sequential displacement of preexisting base pairings involving successive small energy barriers. The proposed mechanism might provide new insight into physical processes during small RNA-mediated gene silencing, which is essential to selection of a target mRNA segment for siRNA design.
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Affiliation(s)
- Jandi Kim
- Department of Biotechnology, Yonsei University, Shinchon-Dong 134, Seodaemun-Gu, Seoul 120-749, Korea
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Gredell JA, Dittmer MJ, Wu M, Chan C, Walton SP. Recognition of siRNA asymmetry by TAR RNA binding protein. Biochemistry 2010; 49:3148-55. [PMID: 20184375 PMCID: PMC2851484 DOI: 10.1021/bi902189s] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
The recognition of small interfering RNAs (siRNAs) by the RNA-induced silencing complex (RISC) and its precursor, the RISC loading complex (RLC), is a key step in the RNA interference pathway that controls the subsequent sequence-specific mRNA degradation. In Drosophila, selection of the guide strand has been shown to be mediated by RLC protein R2D2, which senses the relative hybridization stability between the two ends of the siRNA. A protein with similar function has yet to be conclusively identified in humans. We show here that human TAR RNA binding protein (TRBP) alone can bind siRNAs in vitro and sense their asymmetry. We also show that TRBP can bind 21-nucleotide single-stranded RNAs, though with far lower affinity than for double-stranded siRNA, and that TRBP cross-links preferentially to the 3'-ends of the guide strands of siRNAs. This suggests that TRBP binding depends both on the sequences of the siRNA strands and on the relative hybridization stability of the ends of the duplex. Together, these results demonstrate the importance of the siRNA-TRBP interaction in the selection of the siRNA guide strand in RNAi.
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Affiliation(s)
- Joseph A. Gredell
- Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824-1226, USA
| | - Michael J. Dittmer
- Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824-1226, USA
| | - Ming Wu
- Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824-1226, USA
| | - Christina Chan
- Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824-1226, USA
| | - S. Patrick Walton
- Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824-1226, USA
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Abstract
The ability to manipulate the RNA interference (RNAi) machinery to specifically silence the expression of target genes could be a powerful therapeutic strategy. Since the discovery that RNAi can be triggered in mammalian cells by short double-stranded RNAs (small interfering RNA, siRNA), there has been a tremendous push by researchers, from academia to big pharma, to move siRNAs into clinical application. The challenges facing siRNA therapeutics are significant. The inherent properties of siRNAs (polyanionic, vulnerable to nuclease cleavage) make clinical application difficult due to poor cellular uptake and rapid clearance. Side effects of siRNAs have also proven to be a further complication. Fortunately, numerous chemical modification strategies have been identified that allow many of these obstacles to be overcome. This unit will present an overview of (1) the chemical modifications available to the nucleic acid chemist for modifying siRNAs, (2) the application of chemical modifications to address specific therapeutic obstacles, and (3) the factors that must be considered when assessing the activity of modified siRNAs.
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Klein-Marcuschamer D, Yadav VG, Ghaderi A, Stephanopoulos GN. De Novo metabolic engineering and the promise of synthetic DNA. ADVANCES IN BIOCHEMICAL ENGINEERING/BIOTECHNOLOGY 2010; 120:101-131. [PMID: 20186529 DOI: 10.1007/10_2009_52] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
The uncertain price and tight supply of crude oil and the ever-increasing demand for clean energy have prompted heightened attention to the development of sustainable fuel technologies that ensure continued economic development while maintaining stewardship of the environment. In the face of these enormous challenges, biomass has emerged as a viable alternative to petroleum for the production of energy, chemicals, and materials owing to its abundance, inexpensiveness, and carbon-neutrality. Moreover, the immense ease and efficiency of biological systems at converting biomass-derived feedstocks into fuels, chemicals, and materials has generated renewed interest in biotechnology as a replacement for traditional chemical processes. Aided by the ever-expanding repertoire of microbial genetics and plant biotechnology, improved understanding of gene regulation and cellular metabolism, and incessantly accumulating gene and protein data, scientists are now contemplating engineering microbial cell factories to produce fuels, chemical feedstocks, polymers and pharmaceuticals in an economically and environmentally sustainable way. This goal resonates with that of metabolic engineering - the improvement of cellular properties through the intelligent design, rational modification, or directed evolution of biochemical pathways, and arguably, metabolic engineering seems best positioned to achieve the concomittant goals of environmental stewardship and economic prolificity.Improving a host organism's cellular traits and the potential design of new phenotypes is strongly dependent on the ability to effectively control the organism's genetic machinery. In fact, finely-tuned gene expression is imperative for achieving an optimal balance between pathway expression and cell viability, while avoiding cytotoxicity due to accumulation of certain gene products or metabolites. Early attempts to engineer a cell's metabolism almost exclusively relied on merely deleting or over-expressing single or multiple genes using recombinant DNA, and intervention targets were predominantly selected based on knowledge of the stoichiometry, kinetics, and regulation of the pathway of interest. However, the distributive nature of metabolic control, as opposed to the existence of a single rate-limiting step, predicates the controlled expression of multiple enzymes in several coordinated pathways to achieve the desired flux, and, as such, simple strategies involving either deleting or over-expressing genes are greatly limited in this context. On the other hand, the use of synthetic or modified promoters, riboswitches, tunable intergenic regions, and translation modulators such as internal ribosome entry sequences, upstream open reading frames, optimized mRNA secondary structures, and RNA silencing have been shown to be enormously conducive to achieving the fine-tuning of gene expression. These modifications to the genetic machinery of the host organism can be best achieved via the use of synthetic DNA technology, and the constant improvement in the affordability and quality of oligonucleotide synthesis suggests that these might well become the mainstay of the metabolic engineering toolbox in the years to come. The possibilities that arise with the use of synthetic oligonucleotides will be delineated herein.
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Affiliation(s)
- Daniel Klein-Marcuschamer
- Bioinformatics and Metabolic Engineering Laboratory, Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
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Kini HK, Walton SP. Effect of siRNA terminal mismatches on TRBP and Dicer binding and silencing efficacy. FEBS J 2009; 276:6576-85. [PMID: 19811537 DOI: 10.1111/j.1742-4658.2009.07364.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
To enhance silencing and avoid off-target effects, siRNAs are often designed with an intentional bias to ensure that the end of the siRNA that contains the guide strand 5' end is less stably hybridized relative to the end containing the passenger strand 5' end. One means by which this is accomplished is to introduce a terminal mismatch, typically by changing the passenger strand sequence to impair its hybridization with the guide strand 5' end. However, there are conflicting reports about the influence of terminal mismatches on the silencing efficacy of siRNAs. Here, the silencing efficiency of siRNAs with a terminal mismatch generated either by altering the guide strand (at the 5' end, nucleotide 1) or the passenger strand (nucleotide 19 from the 5' end) was examined. Subsequently, we studied the relationship between the silencing efficiency of the siRNAs and their binding to the RNA-induced silencing complex loading complex proteins HIV transactivating response RNA-binding protein and Dicer in H1299 cytoplasmic extracts. Binding of siRNA and the transactivating response RNA-binding protein was significantly reduced by terminal mismatches, which largely agrees with the reduction in eventual silencing efficacy of the siRNAs. Single terminal mismatches led to a small increase in Dicer binding, as expected, but this did not lead to an improvement in silencing activity. These results demonstrate that introduction of mismatches to control siRNA asymmetry may not always improve target silencing, and that care should be taken when designing siRNAs using this technique.
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Affiliation(s)
- Hemant K Kini
- Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824-1226, USA
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43
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Andreeva Z, Ho AYY, Barthet MM, Potocký M, Bezvoda R, Žárský V, Marc J. Phospholipase D family interactions with the cytoskeleton: isoform delta promotes plasma membrane anchoring of cortical microtubules. FUNCTIONAL PLANT BIOLOGY : FPB 2009; 36:600-612. [PMID: 32688673 DOI: 10.1071/fp09024] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/23/2009] [Accepted: 04/25/2009] [Indexed: 05/06/2023]
Abstract
Phospholipase D (PLD) is a key enzyme in signal transduction - mediating plant responses to various environmental stresses including drought and salinity. Isotype PLDδ interacts with the microtubule cytoskeleton, although it is unclear if, or how, each of the 12 PLD isotypes in Arabidopsis may be involved mechanistically. We employed RNA interference in epidermal cells of Allium porrum L. (leek) leaves, in which the developmental reorientation of cortical microtubule arrays to a longitudinal direction is highly sensitive to experimental manipulation. Using particle bombardment and transient transformation with synthetic siRNAs targeting AtPLDα, β, γ, δ, ॉ and ζ, we examined the effect of 'cross-target' silencing orthologous A. porrum genes on microtubule reorientation dynamics during cell elongation. Co-transformation of individual siRNAs together with a GFP-MBD microtubule-reporter gene revealed that siRNAs targeting AtPLDδ promoted, whereas siRNAs targeting AtPLDβ and γ reduced, longitudinal microtubule orientation in A. porrum. These PLD isotypes, therefore, interact, directly or indirectly, with the cytoskeleton and the microtubule-plasma membrane interface. The unique response of PLDδ to silencing, along with its exclusive localisation to the plasma membrane, indicates that this isotype is specifically involved in promoting microtubule-membrane anchorage.
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Affiliation(s)
- Zornitza Andreeva
- School of Biological Sciences, Macleay Building A12, University of Sydney, Sydney, NSW 2006, Australia
| | - Angela Y Y Ho
- School of Biological Sciences, Macleay Building A12, University of Sydney, Sydney, NSW 2006, Australia
| | - Michelle M Barthet
- School of Biological Sciences, Macleay Building A12, University of Sydney, Sydney, NSW 2006, Australia
| | - Martin Potocký
- Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6, Czech Republic
| | - Radek Bezvoda
- Department of Plant Physiology, Faculty of Science, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic
| | - Viktor Žárský
- Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6, Czech Republic
| | - Jan Marc
- School of Biological Sciences, Macleay Building A12, University of Sydney, Sydney, NSW 2006, Australia
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Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica. BMC Microbiol 2009; 9:38. [PMID: 19222852 PMCID: PMC2652455 DOI: 10.1186/1471-2180-9-38] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2008] [Accepted: 02/17/2009] [Indexed: 12/15/2022] Open
Abstract
Background Entamoeba histolytica is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in Entamoeba histolytica. Results An episomal vector-based system, using the E. histolytica U6 promoter to drive expression of 29-basepair short hairpin RNAs, was developed to target protein-encoding genes in E. histolytica. The short hairpin RNAs successfully knocked down protein levels of all three unrelated genes tested with this system: Igl, the intermediate subunit of the galactose- and N-acetyl-D-galactosamine-inhibitable lectin; the transcription factor URE3-BP; and the membrane binding protein EhC2A. Igl levels were reduced by 72%, URE3-BP by 89%, and EhC2A by 97%. Conclusion Use of the U6 promoter to drive expression of 29-basepair short hairpin RNAs is effective at knocking down protein expression for unrelated genes in Entamoeba histolytica, providing a useful tool for the study of this parasite.
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Leonard JN, Shah PS, Burnett JC, Schaffer DV. HIV evades RNA interference directed at TAR by an indirect compensatory mechanism. Cell Host Microbe 2008; 4:484-94. [PMID: 18996348 PMCID: PMC2742160 DOI: 10.1016/j.chom.2008.09.008] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2007] [Revised: 08/11/2008] [Accepted: 09/08/2008] [Indexed: 02/06/2023]
Abstract
HIV can rapidly evolve when placed under selective pressure, including immune surveillance or the administration of antiretroviral drugs. Typically, a variant protein allows HIV to directly evade the selective pressure. Similarly, HIV has escaped suppression by RNA interference (RNAi) directed against viral RNAs by acquiring mutations at the target region that circumvent RNAi-mediated inhibition while conserving necessary viral functions. However, when we directed RNAi against the viral TAR hairpin, which plays an indispensable role in viral transcription, resistant strains were recovered, but none carried a mutation at the target site. Instead, we isolated several strains carrying promoter mutations that indirectly compensated for the RNAi by upregulating viral transcription. Combining RNAi with the application of an antiviral drug blocked replication of such mutants. Evolutionary tuning of viral transcriptional regulation may serve as a general evasion mechanism that may be targeted to improve the efficacy of antiviral therapy.
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Affiliation(s)
- Joshua N Leonard
- Department of Chemical Engineering and the Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA 94720, USA
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