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The Power of Silence: Application of Small Interfering RNAs to Gastrointestinal Diseases

2007; Elsevier BV; Volume: 132; Issue: 7 Linguagem: Inglês

10.1053/j.gastro.2007.04.056

1528-0012

Ella H. Sklan, Jeffrey S. Glenn,

RNA modifications and cancer

The exciting gene-silencing technology that is finding widespread applications in biology, and offering tantalizing prospects in medicine, was first described in plants and worms as a defense mechanism against invading pathogens.1Hamilton A.J. Baulcombe D.C. A species of small antisense RNA in posttranscriptional gene silencing in plants.Science. 1999; 286: 950Crossref PubMed Scopus (2382) Google Scholar RNA interference (RNAi) is a naturally occurring cellular mechanism wherein foreign double-stranded RNA introduced into the cell induces posttranscriptional gene silencing of RNA molecules with sequence similarity to the introduced RNA. Importantly, synthetic double-stranded RNA molecules with the appropriate characteristics designed against an RNA target of interest can exploit this process to result in the degradation of virtually any targeted RNA. The efficiency and specificity of this process have revolutionized in vitro studies, thereby permitting an almost instant means to “silence” or decrease the expression of any gene function. If this same technology can be practically adapted to clinical use, it will similarly revolutionize medical practice. The relative ease of accessing various parts of the gastrointestinal (GI) tract may render selected GI disorders particularly amenable to this technology. After a brief review of the mechanism of RNA silencing, a short summary of the main advantages and disadvantages of this technology and a survey of various potential GI targets are presented. Double-stranded RNAs in the cell’s cytoplasm—usually a hallmark of RNA virus replicative intermediates—can be recognized and cleaved by the RNAase Dicer into 21- to 25-bp-long RNA fragments with overhangs of 2 nucleotides on both sides, initiating the RNAi response (Figure 1). Synthetic short interfering RNAs (siRNAs) prepared with the correct length and desired sequence can be delivered to the cytoplasm and enter the pathway at this stage. Similarly, short “hairpin RNA” (shRNA) molecules wherein one end of a short RNA duplex is joined by an intervening small stretch of RNA, can be produced within the cell as part of a vector-encoded RNA transcript and further cleaved by Dicer to generate siRNA.2Carmell M.A. Hannon G.J. RNase III enzymes and the initiation of gene silencing.Nat Struct Mol Biol. 2004; 11: 214Crossref PubMed Scopus (320) Google Scholar The expression and silencing efficiency of this latter approach can be greatly enhanced by incorporating natural microRNA sequences into the transcript, so-called shRNAmir.3Chang K. Elledge S.J. Hannon G.J. Lessons from Nature: microRNA-based shRNA libraries.Nat Meth. 2006; 3: 707Crossref PubMed Scopus (228) Google Scholar However generated, the 2 strands of the siRNA molecule are then incorporated into the RNA-induced silencing complex (RISC) and separated. The RISC retains the antisense siRNA strand (guide strand). Together with the cleavage enzyme argonaute-2 (AGO2) and other cellular factors, the silencing complex is now activated and searches for complementary target mRNA sequences. Once identified, the target is cleaved by AGO2 and the RISC is recycled to cleave another mRNA target.4Rana T.M. Illuminating the silence: understanding the structure and function of small RNAs.Nat Rev Mol Cell Biol. 2007; 8: 23Crossref PubMed Scopus (870) Google Scholar siRNAs can be synthesized directly against their target with no need for expensive and time-consuming drug development. They are highly specific (can target only a diseased allele) and can be prepared against every gene whose sequence is known using the same production platform. Despite these advantages, there are still major obstacles to overcome before the successful use of siRNA technology for clinical applications.5Jackson A.L. Burchard J. Leake D. Reynolds A. Schelter J. Guo J. Johnson J.M. Lim L. Karpilow J. Nichols K. Marshall W. Khvorova A. Linsley P.S. Position-specific chemical modification of siRNAs reduces ”off-target” transcript silencing.RNA. 2006; 12: 1197Crossref PubMed Scopus (671) Google Scholar, 6Jackson A.L. Burchard J. Schelter J. Chau B.N. Cleary M. Lim L. Linsley P.S. Widespread siRNA ”off-target” transcript silencing mediated by seed region sequence complementarity.RNA. 2006; 12: 1179Crossref PubMed Scopus (762) Google Scholar Standard siRNAs are poorly suited to act as systemic drugs owing to their short half-life in serum, caused by rapid degradation by serum nucleases.7Soutschek J. Akinc A. Bramlage B. Charisse K. Constien R. Donoghue M. Elbashir S. Geick A. Hadwiger P. Harborth J. John M. Kesavan V. Lavine G. Pandey R.K. Racie T. Rajeev K.G. Rohl I. Toudjarska I. Wang G. Wuschko S. Bumcrot D. Koteliansky V. Limmer S. Manoharan M. Vornlocher H.-P. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.Nature. 2004; 432: 173Crossref PubMed Scopus (1968) Google Scholar, 8Haupenthal J. Baehr C. Kiermayer S. Zeuzem S. Piiper A. Inhibition of RNAse A family enzymes prevents degradation and loss of silencing activity of siRNAs in serum.Biochem Pharmacol. 2006; 71: 702Crossref PubMed Scopus (126) Google Scholar Various chemical modifications can increase serum stability,9Elmén J. Thonberg H. Ljungberg K. Frieden M. Westergaard M. Xu Y. Wahren B. Liang Z. Ørum H. Koch T. Wahlestedt C. Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality.Nucl Acids Res. 2005; 33: 439-447Crossref PubMed Scopus (481) Google Scholar, 10Czauderna F. Fechtner M. Dames S. Aygun H. Klippel A. Pronk G.J. Giese K. Kaufmann J. Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells.Nucl Acids Res. 2003; 31: 2705-2716Crossref PubMed Scopus (552) Google Scholar, 11Braasch D.A. Jensen S. Liu Y. Kaur K. Arar K. White M.A. Corey D.R. 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Demeneix B.A. Lipid-mediated siRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels.J Gene Med. 2005; 7: 198Crossref PubMed Scopus (168) Google Scholar coupling of the siRNA passenger strand to cholesterol,7Soutschek J. Akinc A. Bramlage B. Charisse K. Constien R. Donoghue M. Elbashir S. Geick A. Hadwiger P. Harborth J. John M. Kesavan V. Lavine G. Pandey R.K. Racie T. Rajeev K.G. Rohl I. Toudjarska I. Wang G. Wuschko S. Bumcrot D. Koteliansky V. Limmer S. Manoharan M. Vornlocher H.-P. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.Nature. 2004; 432: 173Crossref PubMed Scopus (1968) Google Scholar encapsulation into polyethylene glycol liposomes,15Morrissey D.V. Lockridge J.A. Shaw L. Blanchard K. Jensen K. Breen W. Hartsough K. Machemer L. Radka S. Jadhav V. Vaish N. Zinnen S. Vargeese C. Bowman K. Shaffer C.S. Jeffs L.B. Judge A. MacLachlan I. Polisky B. Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs.Nat Biotech. 2005; 23: 1002Crossref PubMed Scopus (1029) Google Scholar or mixing siRNAs with a fusion protein consisting of the RNA-binding protein protamine and a targeting Fab antibody fragment.16Vornlocher H.-P. Antibody-directed cell-type-specific delivery of siRNA.Trends Mol Med. 2006; 12: 1Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Those modifications have to be specifically matched to suit the target organ. Some of these applications may be limited by immunogenicity. Although delivery is a major issue in the use of siRNAs as therapeutic drugs, the problems do not end once the siRNA enters the cell. Microarray profiling experiments have shown that siRNA-mediated silencing may be less specific than originally believed, with numerous off-target effects,17Jackson A.L. Bartz S.R. Schelter J. Kobayashi S.V. Burchard J. Mao M. Li B. Cavet G. Linsley P.S. Expression profiling reveals off-target gene regulation by RNAi.Nat Biotech. 2003; 21: 635Crossref PubMed Scopus (1949) Google Scholar, 18Chi J.-T. Chang H.Y. Wang N.N. Chang D.S. Dunphy N. Brown P.O. Genomewide view of gene silencing by small interfering RNAs.Proc Natl Acad Sci. 2003; 100: 6343-6346Crossref PubMed Scopus (270) Google Scholar, 19Semizarov D. Frost L. Sarthy A. Kroeger P. Halbert D.N. Fesik S.W. Specificity of short interfering RNA determined through gene expression signatures.Proc Natl Acad Sci U S A. 2003; 100: 6347-6352Crossref PubMed Scopus (449) Google Scholar although these can be somewhat mitigated by modifying the siRNAs to decrease partial complementarity with the off-target sequences. Although siRNAs were originally thought to be too short to induce immunologic response, several reports now show that a substantial number of shRNA vectors can trigger an interferon response.20Bridge A.J. Pebernard S. Ducraux A. Nicoulaz A.-L. Iggo R. Induction of an interferon response by RNAi vectors in mammalian cells.Nat Genet. 2003; 34: 263Crossref PubMed Scopus (860) Google Scholar, 21Sledz C.A. Holko M. de Veer M.J. Silverman R.H. Williams B.R.G. Activation of the interferon system by short-interfering RNAs.Nat Cell Biol. 2003; 5: 834Crossref PubMed Scopus (1232) Google Scholar Another previously overlooked aspect is interference with the processing and function of endogenous microRNAs. The latter are highly conserved small RNA molecules encoded in host cell genomes that typically bind to the 3′-untranslated regions (3′-UTR) of specific mRNAs and are increasingly recognized as important regulators of normal gene expression and differentiation.22Nilsen T.W. Mechanisms of microRNA-mediated gene regulation in animal cells.Trends Genet. 2007; 23: 249Abstract Full Text Full Text PDF Scopus (502) Google Scholar MicroRNA and siRNA pathways share several cellular components and high expression levels of microRNA-like shRNAs, in a gene therapy vector was recently shown to cause major toxicity in mice, inducing liver injury and death, presumably due to oversaturation of endogenous small RNA pathways.23Grimm D. Streetz K.L. Jopling C.L. Storm T.A. Pandey K. Davis C.R. Marion P. Salazar F. Kay M.A. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.Nature. 2006; 441: 537-541Crossref PubMed Scopus (1407) Google Scholar Despite these challenges, there have been numerous attempts to explore the potential of applying siRNA technology to a growing list of digestive diseases (Figure 2). Most of the work in the GI system has employed siRNAs targeting the liver. In fact, the first paper to show that siRNAs can protect from disease in a whole animal used siRNA directed against the Fas gene (involved in mediating apopotosis) in mouse experimental fulminant hepatitis. siRNA-mediated Fas inhibition prevented hepatocyte death and prolonged survival.24Song E. Lee S.-K. Wang J. Ince N. Ouyang N. Min J. Chen J. Shankar P. Lieberman J. RNA interference targeting Fas protects mice from fulminant hepatitis.Nat Med. 2003; 9: 347Crossref PubMed Scopus (1036) Google Scholar Targeting viral RNA genomes or replicative intermediates with siRNA has shown efficacy against several hepatitis viruses. siRNAs targeting various sites in the hepatitis A virus (HAV) nonstructural genes efficiently inhibited HAV replication. Consecutive siRNA transfections resulted, however, in the emergence of viral escape against most of siRNA sequences.25Kusov Y. Kanda T. Palmenberg A. Sgro J.-Y. Gauss-Muller V. Silencing of hepatitis A virus infection by small interfering RNAs.J Virol. 2006; 80: 5599-5610Crossref PubMed Scopus (49) Google Scholar Thus, as might be expected, siRNA-based therapies are prone to the development of resistance. The design of siRNA molecules against essential regulatory RNA sequences might be one way of dealing with this. siRNA inhibition of hepatitis B virus (HBV) replication has been the subject of many studies.26Klein C. Bock C.T. Wedemeyer H. Wustefeld T. Locarnini S. Dienes H.P. Kubicka S. Manns M.P. Trautwein C. Inhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA.Gastroenterology. 2003; 125: 9Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 27Peng J. Zhao Y. Mai J. Pang W.K. Wei X. Zhang P. Xu Y. Inhibition of hepatitis B virus replication by various RNAi constructs and their pharmacodynamic properties.J Gen Virol. 2005; 86: 3227Crossref PubMed Scopus (23) Google Scholar, 28Xuan B. Qian Z. Hong J. Huang W. EsiRNAs inhibit hepatitis B virus replication in mice model more efficiently than synthesized siRNAs.Virus Res. 2006; 118: 150Crossref PubMed Scopus (21) Google Scholar, 29Giladi H. Ketzinel-Gilad M. Rivkin L. Felig Y. Nussbaum O. Galun E. Small interfering RNA inhibits hepatitis B virus replication in mice.Mol Ther. 2003; 8: 769-776Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar Cotransfection of recombinant adenoviruses expressing HBV-specific shRNAs with HBV-encoding plasmids suppressed HBV gene expression and replication.30McCaffrey A.P. Nakai H. Pandey K. Huang Z. Salazar F.H. Xu H. Wieland S.F. Marion P.L. Kay M.A. Inhibition of hepatitis B virus in mice by RNA interference.Nat Biotechnol. 2003; 21: 630-639Crossref Scopus (593) Google Scholar Uprichard et al31Uprichard S.L. Boyd B. Althage A. Chisari F.V. Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs.Proc Natl Acad Sci U S A. 2005; 102: 773-778Crossref PubMed Scopus (184) Google Scholar used similar vectors to show that preexisting viral replication in HBV transgenic mice could be inhibited to almost undetectable levels for at least 26 days. Morrissey et al,15Morrissey D.V. Lockridge J.A. Shaw L. Blanchard K. Jensen K. Breen W. Hartsough K. Machemer L. Radka S. Jadhav V. Vaish N. Zinnen S. Vargeese C. Bowman K. Shaffer C.S. Jeffs L.B. Judge A. MacLachlan I. Polisky B. Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs.Nat Biotech. 2005; 23: 1002Crossref PubMed Scopus (1029) Google Scholar using a liposome formulation, achieved about a 1.0 log10 reduction in serum HBV DNA that was stable for 6 weeks with weekly dosing. There have been a variety of attempts to demonstrate proof-of-concept that the HCV genome might be susceptible to siRNA-mediated inhibition. siRNAs targeted to the HCV 5′-UTR successfully inhibited HCV replication in a transient replication model.32Prabhu R. Vittal P. Yin Q. Flemington E. Garry R. Robichaux W.H. Dash S. Small interfering RNA effectively inhibits protein expression and negative strand RNA synthesis from a full-length hepatitis C virus clone.J Med Virol. 2005; 76: 511-519Crossref PubMed Scopus (42) Google Scholar Efficient inhibition of HCV IRES-mediated expression from 6 different genotypes has also been demonstrated.33Prabhu R. Garry R.F. Dash S. Small interfering RNA targeted to stem-loop II of the 5′ untranslated region effectively inhibits expression of six HCV genotypes.Virol J. 2006; 3: 100Crossref PubMed Scopus (43) Google Scholar, 30McCaffrey A.P. Nakai H. Pandey K. Huang Z. Salazar F.H. Xu H. Wieland S.F. Marion P.L. 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Susceptibility of human hepatitis delta virus RNAs to small interfering RNA action.J Virol. 2003; 77: 9728-9731Crossref PubMed Scopus (57) Google Scholar During HDV replication, 3 RNA species accumulate: the genome, its exact complement (the anti-genome), and an 800-nt polyadenylated mRNA (of the same polarity as the antigenome and encoding the delta antigen proteins). Only siRNA sequences targeted against HDV mRNA sequences interfered with HDV genome replication. Genomic and antigenomic RNAs are probably resistant owing to their nuclear localization, making them inaccessible to siRNA-mediated degradation. Another antiviral approach is to inhibit host genes upon which viruses depend for their replication. A key advantage here is that the targeted locus is not under genetic control of the virus. Thus, simple viral mutagenesis cannot readily lead to the development of resistance. siRNA-mediated silencing of La, PTB, hVAP-33, and a rab-GAP protein—all of which are cellular cofactors for HCV—markedly diminished expression of the targeted endogenous genes, and substantially blocked HCV replication in Huh-7 cells.39Zhang J. Yamada O. Sakamoto T. Yoshida H. Iwai T. Matsushita Y. Shimamura H. Araki H. Shimotohno K. Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus.Virology. 2004; 320: 135Crossref PubMed Scopus (81) Google Scholar, 40Sklan E, Staschke K, Myers T, Glenn J. A Rab-GAP TBC domain protein binds hepatitis C virus NS5A and mediates viral replication. (submitted).Google Scholar Retroviral vectors expressing shRNAs against proteasome α-subunit 7 or Hu antigen R yielded similar inhibition of HCV subgenomic replication.41Korf M. Jarczak D. Beger C. Manns M.P. Kruger M. Inhibition of hepatitis C virus translation and subgenomic replication by siRNAs directed against highly conserved HCV sequence and cellular HCV cofactors.J Hepatol. 2005; 43: 225Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar The existence of backup mechanisms available to the host cell, but not the virus, presumably affords the potential for a therapeutic index by allowing the cell to survive via other functionally compensating host gene products. A key advantage of siRNA is its versatility in adaptation to new targets. Thus, as specific genes become implicated in the pathogenesis of a given disease, they can be readily targeted by siRNA. Examples of this are found in siRNA technology’s direction at a broad array of GI tumor targets. Although most are restricted to in vitro systems or preclinical in vivo models, they provide a glimpse of their potential to impact as either monotherapies or adjuvant therapies of the future. siRNAs against genes involved in the development of esophageal cancer were evaluated. Three such studies using siRNAs against survivin (a member of inhibitors of apoptosis regulator family42Wang Y. Zhu H. Quan L. Zhou C. Bai J. Zhang G. Zhan Q. Xu N. Downregulation of survivin by RNAi inhibits the growth of esophageal carcinoma cells.Cancer Biol Ther. 2005; 4: 974-978Crossref PubMed Scopus (137) Google Scholar), osteopontin (an integrin binding secreted adhesive glycoprotein43Ito T. Hashimoto Y. Tanaka E. Kan T. Tsunoda S. Sato F. Higashiyama M. Okumura T. Shimada Y. 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EphA2: a determinant of malignant cellular behavior and a potential therapeutic target in pancreatic adenocarcinoma.Oncogene. 2004; 23: 1448Crossref PubMed Scopus (202) Google Scholar Similarly, treatment with siRNA against VAV1, a Rho GTPase family guanine nucleotide exchange factor associated with decreased survival of pancreatic cancer patients,54Fernandez-Zapico M.E. Gonzalez-Paz N.C. Weiss E. Savoy D.N. Molina J.R. Fonseca R. Smyrk T.C. Chari S.T. Urrutia R. Billadeau D.D. Ectopic expression of VAV1 reveals an unexpected role in pancreatic cancer tumorigenesis.Cancer Cell. 2005; 7: 39Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar abrogated neoplastic cellular proliferation in vitro and in nude mice. Highlighting the selectivity achievable with siRNA technology, siRNA specifically directed against mutant forms of the K-ras gene implicated in the development of pancreatic adenocarcinoma, resulted in alteration of tumor cell behavior.55Fleming J.B. Shen G.-L. Holloway S.E. Davis M. Brekken R.A. Molecular consequences of silencing mutant K-ras in pancreatic cancer cells: justification for K-ras-directed therapy.Mol Cancer Res. 2005; 3: 413Crossref PubMed Scopus (166) Google Scholar Genes involved in the development and progression of colon cancer have been described in elegant detail, and new ones are being uncovered. Many of these genes can be successfully inhibited by siRNA, including targets such as β-catenin,56Verma U.N. Surabhi R.M. Schmaltieg A. Becerra C. Gaynor R.B. Small interfering RNAs directed against β-catenin inhibit the in vitro and in vivo growth of colon cancer cells.Clin Cancer Res. 2003; 9: 1291-1300PubMed Google Scholar and the RON (recepteur d’origine nantais) receptor tyrosine kinase,57Xu X.-M. Wang D. Shen Q. Chen Y.-Q. Wang M.-H. 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Suppression of progression and metastasis of established colon tumors in mice by intravenous delivery of short interfering RNA targeting KITENIN, a metastasis-enhancing protein.Cancer Res. 2005; 65: 8993Crossref PubMed Scopus (50) Google Scholar and the oncogene murine double minute 2 (mdm2).61Yu Y. Sun P. Sun L.-c Liu G.-y Chen G.-h Shang L.-h Wu H.-b Hu J. Li Y. Mao Y.-l Sui G.-j Sun X.-w Downregulation of MDM2 expression by RNAi inhibits LoVo human colorectal adenocarcinoma cells growth and the treatment of LoVo cells with mdm2siRNA3 enhances the sensitivity to cisplatin.Biochem Biophys Res Commun. 2006; 339: 71Crossref PubMed Scopus (12) Google Scholar In many of these strategies, sufficient delivery to the target sites without excessive inhibition of these same pathways in healthy cells represents an incompletely resolved challenge. In addition, it is also unlikely that depleting a single gene will be sufficient to effectively treat cancer. An effective delivery strategy for one siRNA, however, should be readily adaptable to cocktails targeting specific collections of relevant genes. Similar approaches might be contemplated for premalignant lesions, such as those associated with Barrett’s esophagus. Similarly, using a murine model of inflammatory bowel disease, Zhang et al62Zhang Y. Cristofaro P. Silbermann R. Pusch O. Boden D. Konkin T. Hovanesian V. Monfils P.R. Resnick M. Moss S.F. Ramratnam B. Engineering mucosal RNA interference in vivo.Mol Ther. 2006; 14: 336Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar found that the rectal application of liposomal siRNA formulations targeting tumor necrosis factor-α led to relative mucosal resistance to experimental colitis. Those formulations were nontoxic and did not elicit an interferon response, providing a means for genetically manipulating mucosal surfaces in vivo. The ability to achieve local delivery via endoscopic application may allow certain GI lesions to be effectively targeted despite current limitations of existing siRNA technology. Ultimately, efficient systemic delivery remains a dominant challenge to widespread clinical use of siRNA technology, the solution to which will have ramifications beyond GI disorders.