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Lewin AS, Smith WC. Gene Therapy for Rhodopsin Mutations. Cold Spring Harb Perspect Med 2022; 12:a041283. [PMID: 35940643 PMCID: PMC9435570 DOI: 10.1101/cshperspect.a041283] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Mutations in RHO, the gene for rhodopsin, account for a large fraction of autosomal-dominant retinitis pigmentosa (adRP). Patients fall into two clinical classes, those with early onset, pan retinal photoreceptor degeneration, and those who experience slowly progressive disease. The latter class of patients are candidates for photoreceptor-directed gene therapy, while former may be candidates for delivery of light-responsive proteins to interneurons or retinal ganglion cells. Gene therapy for RHO adRP may be targeted to the mutant gene at the DNA or RNA level, while other therapies preserve the viability of photoreceptors without addressing the underlying mutation. Correcting the RHO gene and replacing the mutant RNA show promise in animal models, while sustaining viable photoreceptors has the potential to delay the loss of central vision and may preserve photoreceptors for gene-directed treatments.
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Affiliation(s)
- Alfred S Lewin
- Departments of Molecular Genetics and Microbiology and Ophthalmology, University of Florida College of Medicine, Gainesville, Florida 32610, USA
| | - W Clay Smith
- Departments of Molecular Genetics and Microbiology and Ophthalmology, University of Florida College of Medicine, Gainesville, Florida 32610, USA
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Fukuda M, Kurihara K, Tanaka Y, Deshimaru M. A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing. RNA (NEW YORK, N.Y.) 2012; 18:1735-1744. [PMID: 22798264 PMCID: PMC3425787 DOI: 10.1261/rna.033399.112] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/25/2012] [Accepted: 06/07/2012] [Indexed: 06/01/2023]
Abstract
Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.
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Affiliation(s)
- Masatora Fukuda
- Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-ku, Fukuoka 814-0180, Japan.
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Sullivan JM, Yau EH, Kolniak TA, Sheflin LG, Taggart RT, Abdelmaksoud HE. Variables and strategies in development of therapeutic post-transcriptional gene silencing agents. J Ophthalmol 2011; 2011:531380. [PMID: 21785698 PMCID: PMC3138052 DOI: 10.1155/2011/531380] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2010] [Revised: 02/17/2011] [Accepted: 02/28/2011] [Indexed: 11/24/2022] Open
Abstract
Post-transcriptional gene silencing (PTGS) agents such as ribozymes, RNAi and antisense have substantial potential for gene therapy of human retinal degenerations. These technologies are used to knockdown a specific target RNA and its cognate protein. The disease target mRNA may be a mutant mRNA causing an autosomal dominant retinal degeneration or a normal mRNA that is overexpressed in certain diseases. All PTGS technologies depend upon the initial critical annealing event of the PTGS ligand to the target RNA. This event requires that the PTGS agent is in a conformational state able to support hybridization and that the target have a large and accessible single-stranded platform to allow rapid annealing, although such platforms are rare. We address the biocomplexity that currently limits PTGS therapeutic development with particular emphasis on biophysical variables that influence cellular performance. We address the different strategies that can be used for development of PTGS agents intended for therapeutic translation. These issues apply generally to the development of PTGS agents for retinal, ocular, or systemic diseases. This review should assist the interested reader to rapidly appreciate critical variables in PTGS development and facilitate initial design and testing of such agents against new targets of clinical interest.
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Affiliation(s)
- Jack M. Sullivan
- Department of Ophthalmology, University at Buffalo SUNY, Buffalo, NY 14214, USA
- Department of Pharmacology and Toxicology, University at Buffalo SUNY, Buffalo, NY 14214, USA
- Department of Physiology and Biophysics, University at Buffalo SUNY, Buffalo, NY 14214, USA
- Neuroscience Program, University at Buffalo SUNY, Buffalo, NY 14214, USA
- Ross Eye Institute, University at Buffalo SUNY, Buffalo, NY 14209, USA
- Veterans Administration Western New York Healthcare System, Medical Research, Buffalo, NY 14215, USA
| | - Edwin H. Yau
- Department of Ophthalmology, University at Buffalo SUNY, Buffalo, NY 14214, USA
- Department of Pharmacology and Toxicology, University at Buffalo SUNY, Buffalo, NY 14214, USA
| | - Tiffany A. Kolniak
- Department of Ophthalmology, University at Buffalo SUNY, Buffalo, NY 14214, USA
- Neuroscience Program, University at Buffalo SUNY, Buffalo, NY 14214, USA
| | - Lowell G. Sheflin
- Department of Ophthalmology, University at Buffalo SUNY, Buffalo, NY 14214, USA
- Veterans Administration Western New York Healthcare System, Medical Research, Buffalo, NY 14215, USA
| | - R. Thomas Taggart
- Department of Ophthalmology, University at Buffalo SUNY, Buffalo, NY 14214, USA
| | - Heba E. Abdelmaksoud
- Department of Neuroscience and Physiology, Upstate Medical University, Syracuse, NY 13215, USA
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Carbonell A, Flores R, Gago S. Trans-cleaving hammerhead ribozymes with tertiary stabilizing motifs: in vitro and in vivo activity against a structured viroid RNA. Nucleic Acids Res 2010; 39:2432-44. [PMID: 21097888 PMCID: PMC3064770 DOI: 10.1093/nar/gkq1051] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Trans-cleaving hammerheads with discontinuous or extended stem I and with tertiary stabilizing motifs (TSMs) have been tested previously against short RNA substrates in vitro at low Mg(2+) concentration. However, the potential of these ribozymes for targeting longer and structured RNAs in vitro and in vivo has not been examined. Here, we report the in vitro cleavage of short RNAs and of a 464-nt highly structured RNA from potato spindle tuber viroid (PSTVd) by hammerheads with discontinuous and extended formats at submillimolar Mg(2+). Under these conditions, hammerheads derived from eggplant latent viroid and peach latent mosaic viroid (PLMVd) with discontinuous and extended formats, respectively, where the most active. Furthermore, a PLMVd-derived hammerhead with natural TSMs showed activity in vivo against the same long substrate and interfered with systemic PSTVd infection, thus reinforcing the idea that this class of ribozymes has potential to control pathogenic RNA replicons.
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Affiliation(s)
- Alberto Carbonell
- Instituto de Biología Molecular y Celular de Plantas, UPV-CSIC, Campus Universidad Politécnica de Valencia, Avenida de los Naranjos, 46022 Valencia, Spain
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Fiskaa T, Birgisdottir AB. RNA reprogramming and repair based on trans-splicing group I ribozymes. N Biotechnol 2010; 27:194-203. [PMID: 20219714 DOI: 10.1016/j.nbt.2010.02.013] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
While many traditional gene therapy strategies attempt to deliver new copies of wild-type genes back to cells harboring the defective genes, RNA-directed strategies offer a range of novel therapeutic applications. Revision or reprogramming of mRNA is a form of gene therapy that modifies mRNA without directly changing the transcriptional regulation or the genomic gene sequence. Group I ribozymes can be engineered to act in trans by recognizing a separate RNA molecule in a sequence-specific manner, and to covalently link a new RNA sequence to this separate RNA molecule. Group I ribozymes have been shown to repair defective transcripts that cause human genetic or malignant diseases, as well as to replace transcript sequences by foreign RNA resulting in new cellular functions. This review provides an overview of current strategies using trans-splicing group I ribozymes in RNA repair and reprogramming.
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Affiliation(s)
- Tonje Fiskaa
- RNA and Transcriptomics Group, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway.
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Rayburn ER, Ezell SJ, Zhang R. Recent advances in validating MDM2 as a cancer target. Anticancer Agents Med Chem 2009; 9:882-903. [PMID: 19538162 PMCID: PMC6728151 DOI: 10.2174/187152009789124628] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2008] [Accepted: 05/14/2008] [Indexed: 12/26/2022]
Abstract
The MDM2 oncogene is overexpressed in various human cancers. Its expression correlates with the phenotypes of high-grade, late-stage, and more resistant tumors. The auto-regulatory loop between MDM2 and the tumor suppressor p53 has long been considered the epitome of a rational target for cancer therapy. As such, many novel agents have been generated to interfere with the interaction of the two proteins, which results in the activation of p53. Among these agents are several small molecule inhibitors synthesized based upon the crystal structures of the MDM2-p53 complex. With use of high-throughput screening, several specific and effective agents for inhibition of the protein-protein interaction were discovered. Recent investigations, however, have demonstrated that many proteins regulate the MDM2-p53 interaction, and that MDM2 may have p53-independent oncogenic functions. In order for novel MDM2 inhibitors to be translated to the clinic, it is necessary to obtain a better understanding of the regulation of MDM2 and of the MDM2-p53 interaction. In particular, the implications of various interactions between certain regulator(s) and MDM2/p53 under different circumstances need to be elucidated to determine which pathway(s) represent the best targets for therapy. Targeting both MDM2 itself and regulators of MDM2 and the MDM2-p53 interaction, or use of MDM2 inhibitors in combination with conventional treatments, may improve prospects for tumor eradication.
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Affiliation(s)
- Elizabeth R. Rayburn
- Department of Pharmacology and Toxicology, Division of Clinical Pharmacology, University of Alabama at Birmingham, USA
| | - Scharri J. Ezell
- Department of Pharmacology and Toxicology, Division of Clinical Pharmacology, University of Alabama at Birmingham, USA
| | - Ruiwen Zhang
- Department of Pharmacology and Toxicology, Division of Clinical Pharmacology, University of Alabama at Birmingham, USA
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Abstract
Defining translational research is still a complex task. In oncology, translational research implies using our basic knowledge learnt from in vitro and in vivo experiments to directly improve diagnostic tools and therapeutic approaches in cancer patients. Moreover, the better understanding of human cancer and its use to design more reliable tumor models and more accurate experimental systems also has to be considered a good example of translational research. The identification and characterization of new molecular markers and the discovery of novel targeted therapies are two main goals in colorectal cancer translational research. However, the straightforward translation of basic research findings, specifically into colorectal cancer treatment and vice versa is still underway. In the present paper, a summarized view of some of the new available approaches on colorectal cancer translational research is provided. Pros and cons are discussed for every approach exposed.
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Li J, Stouffs M, Serrander L, Banfi B, Bettiol E, Charnay Y, Steger K, Krause KH, Jaconi ME. The NADPH oxidase NOX4 drives cardiac differentiation: Role in regulating cardiac transcription factors and MAP kinase activation. Mol Biol Cell 2006; 17:3978-88. [PMID: 16775014 PMCID: PMC1556380 DOI: 10.1091/mbc.e05-06-0532] [Citation(s) in RCA: 225] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Reactive oxygen species (ROS) generated by the NOX family of NADPH oxidases have been described to act as second messengers regulating cell growth and differentiation. However, such a function has hitherto not been convincingly demonstrated. We investigated the role of NOX-derived ROS in cardiac differentiation using mouse embryonic stem cells. ROS scavengers prevented the appearance of spontaneously beating cardiac cells within embryoid bodies. Down-regulation of NOX4, the major NOX isoform present during early stages of differentiation, suppressed cardiogenesis. This was rescued by a pulse of low concentrations of hydrogen peroxide 4 d before spontaneous beating appears. Mechanisms of ROS-dependent signaling included p38 mitogen-activated protein kinase (MAPK) activation and nuclear translocation of the cardiac transcription factor myocyte enhancer factor 2C (MEF2C). Our results provide first molecular evidence that the NOX family of NADPH oxidases regulate vertebrate developmental processes.
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Affiliation(s)
- Jian Li
- *Laboratory of Biology of Aging, Department of Rehabilitation and Geriatrics, and
| | - Michael Stouffs
- *Laboratory of Biology of Aging, Department of Rehabilitation and Geriatrics, and
| | - Lena Serrander
- *Laboratory of Biology of Aging, Department of Rehabilitation and Geriatrics, and
| | - Botond Banfi
- *Laboratory of Biology of Aging, Department of Rehabilitation and Geriatrics, and
| | - Esther Bettiol
- *Laboratory of Biology of Aging, Department of Rehabilitation and Geriatrics, and
| | - Yves Charnay
- Division of Neuropsychiatry, Geneva University Hospitals, 1225 Chêne-Bourg, Switzerland; and
| | - Klaus Steger
- Justus-Liebig-University, Institute of Veterinary Anatomy, Histology, and Embryology, Giessen, Germany
| | - Karl-Heinz Krause
- *Laboratory of Biology of Aging, Department of Rehabilitation and Geriatrics, and
| | - Marisa E. Jaconi
- *Laboratory of Biology of Aging, Department of Rehabilitation and Geriatrics, and
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Famulok M, Mayer G. Intramers and aptamers: applications in protein-function analyses and potential for drug screening. Chembiochem 2005; 6:19-26. [PMID: 15637667 DOI: 10.1002/cbic.200400299] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Affiliation(s)
- Michael Famulok
- Rheinische Friedrich-Wilhelms Universität Bonn, Kekulé-Institut für Organische Chemie und Biochemie, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany.
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Wickramasinghe NS, Nagaraj NS, Vigneswaran N, Zacharias W. Cathepsin B promotes both motility and invasiveness of oral carcinoma cells. Arch Biochem Biophys 2005; 436:187-95. [PMID: 15752724 DOI: 10.1016/j.abb.2005.01.023] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2005] [Revised: 01/27/2005] [Indexed: 11/16/2022]
Abstract
We previously demonstrated that overexpression of cathepsin B (CB) protease in oral squamous cell carcinomas correlated positively with advanced tumor stage and poor histologic malignancy grade. Here we examined whether CB contributes to the invasiveness of oral carcinoma cells. For RNA-mediated inhibition, two ribozymes that target CB mRNA were designed and stably expressed in the oral squamous cell carcinoma cell line 1386Tu. Both ribozymes diminished expression of CB mRNA, protein, and activity, without affecting cathepsin D or beta-actin, as determined by quantitative real-time PCR, Western blots, and protease activity assays. Matrigel invasion assays showed that the invasiveness of the cells was significantly reduced by the expressed ribozymes and, surprisingly, the motilities of the ribozyme-transfected cells were also diminished. Our results document a direct role for CB in promoting oral cancer spread and invasion, and open the possibility of controlling oral carcinoma malignancy and metastasis by targeting CB with RNA inhibitor strategies.
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Affiliation(s)
- Nalinie S Wickramasinghe
- Department of Medicine, James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, USA
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Gu ZP, Wang YJ, Wu Y, Li JG, Chen NA. Synthesis of ribozyme against vascular endothelial growth factor 165 and its biological activity in vitro. World J Gastroenterol 2004; 10:1495-8. [PMID: 15133860 PMCID: PMC4656291 DOI: 10.3748/wjg.v10.i10.1495] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To investigate the designation, synthesis and biological activity of against vascular endothelial growth factor165 (VEGF165) ribozyme.
METHODS: The ribozyme against VEGF165 was designed with computer. The transcriptional vector was constructed which included the anti-VEGF165 ribozyme and 5’, 3’ self-splicing ribozymes. The hammerhead ribozyme and substrate VEGF165 mRNA were synthesized through transcription in vitro. The cleavage activity of the ribozyme on target RNA was observed in a cell-free system.
RESULTS: The anti-VEGF165 ribozyme was released properly from the transcription of pGEMRz212 cleaved by 5’ and 3’ self-splicing ribozymes which retained its catalytic activity, and the cleavage efficiency of ribozyme reached 90.7%.
CONCLUSION: The anti-VEGF165 ribozyme designed with computer can cleave VEGF165 mRNA effectively.
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Affiliation(s)
- Zhong-Ping Gu
- Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, Shaanxi Province, China.
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