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Beyond silencing — engineering applications of RNA interference and antisense technology for altering cellular phenotype
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Beyond silencing — engineering applications of RNA interference and antisense technology for altering cellular phenotype Colin G Hebert1,2,4, James J Valdes2 and William E Bentley1,3,4 Since its discovery 10 years ago, RNA interference (RNAi) has evolved from a research tool into a powerful method for altering the phenotype of cells and whole organisms. Its near universal applicability coupled with its pinpoint accuracy for suppressing target proteins has altered the landscape of many fields. While there is considerable intellectual investment in therapeutics, its potential extends far beyond. In this review, we explore some of these emerging applications — metabolic engineering for enhancing recombinant protein production in both insect and mammalian cell systems, antisense technologies in bacteria as next generation antibiotics, and RNAi in plant biotechnology for improving productivity and nutritional value. Addresses 1 Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant Science Building, College Park, MD 20742, USA 2 U.S. Army Edgewood Chemical Biological Center, AMSRD-ECB-AP-B/ Michel E3330, 5183 Black Hawk RD, Aberdeen Proving Ground, MD 21010, USA 3 Department of Chemical and Biomolecular Engineering, University of Maryland College Park, College Park, MD 20742, USA 4 Room 2330, Jeong H. Kim Engineering Building (Building #225), Fischell Department of Bioengineering, University of Maryland College Park, College Park, MD 20742, USA Corresponding author: Bentley, William E ([email protected])
Current Opinion in Biotechnology 2008, 19:500–505 This review comes from a themed issue on Tissue, Cell and Pathyway Engineering Edited by William Bentley and Michael Betenbaugh
0958-1669/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2008.08.006
Introduction Since its discovery in 1998 [1], RNA interference (RNAi) has quickly moved from a tool for single gene loss-offunction studies to a means of implementing genetic changes in a specific manner over a wide range of organisms and cell types. In addition to unraveling the mechanisms, origins, and purpose of RNAi [2], research has focused on improving and creating new delivery methods for both cells and animals [3], as well as genome-wide RNAi screens for functional analysis [4]. RNAi has also been touted as next great therapeutic tool for combating a wide range of diseases, including cancer, neurodegenerative disease, viral infection, and ocular disorders [5,6]. Great potential exists, but difficulties remain, including Current Opinion in Biotechnology 2008, 19:500–505
effectively delivering the RNAi of interest into the target cell and precisely quantifying specific and nonspecific side effects. Given the numerous reviews and commentary recently published regarding the various aspects of RNAi as an in vivo therapeutic tool [5,6], and the many reviews of the basis for its function, these will not be the focus of this article. Rather, this review seeks to give an overview of some of the applications of RNAi and antisense technology as a means of affecting phenotype at the cellular and organismal level. We describe applications relating to bioprocessing (metabolic engineering for enhancing recombinant protein production) as well as interesting approaches for antisense RNA in bacteria, and, finally, RNAi in plant biotechnology (Figure 1).
RNAi in metabolic engineering Metabolic engineering typically involves the rational alteration of a host cell’s genotype (genes and regulatory structures) in order to achieve a desired phenotype [7,8]. In addition to efficient production of the molecule of interest, desirable phenotypes include the ability to survive well in the adverse conditions, such as decreased oxygen and nutrients or increased cellular and oxidative stress normally experienced by cells overproducing protein [9]. In general, this involves altering the central metabolism, seeking to increase the flux through specific pathways while decreasing others. Although this has traditionally been accomplished through the overexpression of certain proteins, a similar approach can be taken using RNAi and other antisense technologies: instead of overexpressing desirable genes, potentially deleterious genes are silenced. RNAi initially found limited use in metabolic engineering, however, perhaps owing to its transient nature, relatively inefficient delivery methods, and the more typical metabolic engineering objective functions that are based on permanent changes in genotype. As RNAi technology has improved by attempting to address issues such as potency, specificity, and stability, several noteworthy examples based on antisense RNA have appeared targeting apoptosis, the cell growth cycle, and glycosylation. Because apoptosis (programmed cell death) can account for up to 80% of cell death in bioreactors and lead to decreased product yield and quality, antiapoptosis engineering has become an important area of metabolic engineering [10]. This strategy has also been implemented using RNAi. Recent examples include RNAi against proteases in CHO [11,12] and Sf9 cells [13] and www.sciencedirect.com
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Figure 1
RNA interference and altering phenotype. RNA constructs are used in research (screening and loss of function), therapeutics [59], metabolic engineering [16], plant biotechnology, and, in single-stranded form, bacteria.
produced by recombinant baculovirus [14]. In each of these examples, the yield of recombinant protein was enhanced as a result of the downregulation of a target protease. Another approach involves altering the cell growth cycle using RNAi. An optimal production cycle is generally biphasic. The cells first grow quickly to a high density before transitioning to a production phase characterized by low growth and high productivity [15]. Achieving this regime is relatively straightforward in bacterial systems, but can be more problematic in eukaryotic cell culture because their state is influenced by the cell cycle. Although past efforts have used more traditional metabolic engineering methods for controlling the cell cycle, recent studies have used RNAi to increase cell growth and protein production in Drosophila S2 cells through the downregulation of cell growth controllers [16,17] and in HEK 293 cells through the downregulation of transcriptional regulators [18]. A final area of metabolic engineering in which RNAi has played a role is glycosylation. Engineering specific glycosylation patterns can increase the potency and stability of therapeutic proteins while at the same time decreasing their immunogenic effects [19]. Several specific examples of using RNAi to fine-tune glycosylation patterns include increasing the antibody-dependent cellular cytotoxicity of antibodies produced in CHO cells through the reduction of a 1,6-fucosyltransferase (FUT8) [20], GDP mannose-4,6-dehydrogenase (GMP) [21], or both FUT8 and GMP [22], as well as improving product quality by reducing sialidase activity in CHO cells [23]. www.sciencedirect.com
As the amount of genomic and metabolomic data increases, additional RNAi-based metabolic engineering targets will surely be identified. Some such screens are already underway [15,24,25], and as more targets are identified, RNAi will continue to expand its influence in the field of metabolic engineering.
Antisense RNA in prokaryotic systems Although the majority of RNA-based cellular engineering has focused on eukaryotic systems, antisense RNA technology has also been employed in bacterial systems, both as a metabolic engineering tool and a potential therapeutic agent. Antisense molecules anneal to complementary mRNA strands, either by blocking translation through steric hindrance or causing rapid degradation, potentially by dsRNA-specific RNases. The efficiency of this disruption depends upon several factors, including the length and structure of the antisense molecule, as well as the intracellular concentration and resistance to the degradation of the target mRNA [26]. In addition to the loss-of-function studies performed in many organisms [26], asRNA has been used as a metabolic engineering tool to enhance the productivity of several bacterial hosts including E. coli [27], Clostridium acetobutylicum [28], and Lactobacillus rhamnosus [29]. Antisense approaches have also been used to protect bacteria against bacteriophages [30]. Another application of asRNA as a means of altering bacterial phenotype is through a reduction in pathogenicity, either by reducing the potency of certain toxins or virulence mechanisms, or through the recovery of antibiotic susceptibility. External guide sequences (EGS), which are small asRNAs expressed from plasmids, bind Current Opinion in Biotechnology 2008, 19:500–505
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complementary mRNA to activate cleavage through the endogenous RNase P activity and have also been used to alter bacterial phenotype. Initial studies used EGS to reduce the levels of alkaline phosphatase and b-galactosidase [31], but further applications in E. coli have restored antibiotic sensitivity in E. coli [32] and decreased host cell invasion rate in Salmonella enterica serovar Typhimurium [33]. Although EGS have been successful in a laboratory setting, their potential as an in vivo therapeutic is somewhat limited because they are generally more effective when expressed on plasmids. As an alternative, many researchers have turned to antisense phosphodiester oligodeoxyribonucleotides (asODNs), which are short, chemically synthesized DNA sequences complementary to the mRNA of the target gene. ODNs have been used to decrease antibiotic resistance in E. coli [34], but have seen limited use directly owing to their rapid degradation by nucleases. Rather, a number of synthetic analogs have been developed, the most promising of which include phosphorothiate ODNs (PS-ODNs), peptide nucleic acids (PNAs), and phosphorodiamidate morpholino oligomers (PMOs) [35–37]. PS-ODNs have been well studied in eukaryotic systems, but have so far seen limited use in bacterial systems, mostly in Mycobacteria. Recent examples include the application of PS-ODNs to inhibit growth in M. tuberculosis [38] and restore antibiotic susceptibility in a methicillin-resistant strain of S. aureus [39]. PNAs have been used to target a number of RNAs in E. coli [40], the Gramnegative human pathogen Klebsiella pneumoniae [41], and S. aureus [42], resulting in inhibited growth, though sometimes at relatively high concentrations. The study involving K. pneumoniae used PNAs targeting two essential genes to cure infected IMR90 cells in culture [41]. Applications involving PMOs have mainly targeted the acyl carrier protein of E. coli [43,44], though efforts are underway to test their ability to silence genes in Bacillus anthrax as well [45]. Despite the success of these synthetic analogs, many challenges remain before they can be successfully applied as antimicrobials, including solubility, binding efficiency, resistance to degradation, and cellular delivery, which is perhaps the most pressing [26]. Antisense technologies have shown great effectiveness in in vitro and cell-free systems; however, they will not emerge as next generation antimicrobial drugs until they can be effectively delivered to their target. As bacteria become increasingly resistant to the current methods of treatment, so increases the importance of developing new antisense technologies.
and antisense RNA against the same gene, dsRNA technology was not applied in plants until 2000, when flower identity genes were silenced used inverted repeats [47]. RNAi technology in plants has been successfully implemented using both intron spliced long dsRNA and hairpin RNAs to silence target genes [48,49]. Chemically inducible vectors have also been developed [50], but are not as practical or safe for large-scale use as tissue-specific or organ-specific promoters for the development of RNAi technology in plants [51]. The main thrust of RNAi application in plants is for the improvement of plant productivity or nutritional value [51]. Several groups have increased the lysine content in corn either through the knockdown of lysine–ketoglutarate reductase/saccharophine dehydrogenase (ZLRK/ SDH), the enzyme responsible for its catabolism [52], or the suppression of specific zein storage proteins [53]. Through the use of an endosperm-specific promoter, Houmard et al. confined the increase in lysine content to the kernels, reducing side effects such as abnormal growth and flower development that can be present in plants overproducing lysine in all tissues [52]. Because lysine is one of the essential amino acids, its introduction into corn has worldwide implications in many western countries where it is used mainly as a livestock feed (and is currently supplemented with lysine) as well as in developing countries, where corn is staple crop for humans. Another application of RNAi to an agriculturally important crop is the development of cottonseed with a reduced amount of gossypol toxin [54]. Through the use of a seed-specific promoter, Sunilkumar et al. were able to reduce the amount of gossypol in cottonseeds well below that deemed safe for human consumption by United Nations Food and Agriculture Organization and World Health Organization standards, potentially opening up a new food source for millions of people in the developing world, while maintaining wild-type levels in leaves and other tissues, preserving their function as protection against insects and disease [54]. This approach in cottonseed has laid the groundwork for the development of similar approaches in other potential food sources that can be hampered by their toxicity, including the tropical legume Lathyrus sativus, cassava, and fava beans [51]. Other examples of RNAi technology for the improvement of plant nutritional value include the development of high-fiber wheat [55], as well as a reduction in the allergenicity of peanuts [56]. Two final examples include modifying the profile of morphinan alkaloids in opium poppy [57], as well as modifying rice plant height to increase lodging resistance and decrease potential damage from wind and rain [58].
RNAi in plants Despite the discovery of post-transcriptional gene silencing in 1998 [46] by crossing plants that produced sense Current Opinion in Biotechnology 2008, 19:500–505
The application of RNAi with the potential to impact the greatest number of people may be in plant biotechnology. www.sciencedirect.com
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In many developing countries, people depend upon certain plants not only for income, but also as a means of survival. As global populations and demand for food continue to increase, new agricultural technologies and techniques will be required to meet that demand in a responsible and sustainable manner. Current strategies include improving the productivity or nutritional value of existing food crops or developing new ones. RNAi has emerged as an important tool in both these areas.
Conclusion Although RNAi has received the most attention for its potential as a next generation therapeutic, the alternate applications presented here are no less important. Metabolic engineering via both RNAi and antisense RNA impact the production of not only therapeutic proteins and antibodies, but also biologically based fuels and chemicals, which is a field that will become increasingly important as resources become limited and sustainability becomes a higher priority. Antisense molecules targeting antibiotic resistant and other pathogenic bacteria will also become important as more bacteria evolve resistance to current antibiotic treatments. Finally, the enhancement of plants through the use of RNAi, either as a means of improving current food crops, such as corn and rice, or developing new ones, such as cottonseed, has the potential to impact the lives of millions of people throughout the world. As the fundamental knowledge and associated technology associated with RNAi continues to increase, applications already under study will improve. This is perhaps one of the marks of a great discovery, that its influence has extended well beyond the use for which it was originally intended. It will be interesting to see what additional applications are developed as RNAi enters into its second decade.
Acknowledgements Partial support of this work was provided by the Department of Defense (CREST Fellowship to CGH) and the National Institutes of Health (GM70851-01).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC: Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998, 391:806-811.
2.
Tomari Y, Zamore PD: Perspective: machines for RNAi. Genes Dev 2005, 19:517-529.
3.
Kumar LD, Clarke AR: Gene manipulation through the use of small interfering RNA (siRNA): from in vitro to in vivo applications. Adv Drug Deliv Rev 2007, 59:87-100.
4.
Echeverri CJ: High-throughput RNAi screening in cultured cells: a user’s guide. Nat Rev Genet 2006, 7:373-383.
5.
de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J: Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov 2007, 6:443-453.
www.sciencedirect.com
6.
Dykxhoorn DM, Palliser D, Lieberman J: The silent treatment: siRNAs as small molecule drugs. Gene Therapy 2006, 13:541-552. Recent and comprehensive review of RNAi as a therapeutic. Discusses fundamentals, delivery technologies, and siRNA efficacy against specific diseases. 7.
Bailey JE: Toward a science of metabolic engineering. Science 1991, 252:1668-1675.
8.
Stephanopoulos G, Vallino JJ: Network rigidity and metabolic engineering in metabolite overproduction. Science 1991, 252:1675-1681.
9.
Rao RV, Bredesen DE: Misfolded proteins, endoplasmic reticulum stress and neurodegeneration. Curr Opin Cell Biol 2004, 16:653-663.
10. Majors BS, Betenbaugh MJ, Chiang GG: Links between metabolism and apoptosis in mammalian cells: applications for anti-apoptosis engineering. Metab Eng 2007, 9:317-326. 11. Wong DCF, Wong KTK, Nissom PM, Heng CK, Yap MGS: Targeting early apoptotic genes in batch and fed-batch CHO cell cultures. Biotechnol Bioeng 2006, 95:350-361. See annotation to Ref. [24]. 12. Lai DZ, Fu L, Weng SJ, Qi LQ, Yu CM, Yu T, Wang HT, Chen W: Blocking caspase-3 activity with a U6SnRNA promoter-driven ribozyme enhances survivability of CHO cells cultured in low serum medium and production of interferon-beta. Biotechnol Bioeng 2004, 85:20-28. 13. Lin CC, Hsu JTA, Huang KL, Tang HK, Shu CW, Lai YK: SfCaspase-I-repressed stable cells: resistance to apoptosis and augmentation of recombinant protein production. Biotechnol Appl Biochem 2007, 48:11-19. 14. Kim EJ, Kramer SF, Hebert CG, Valdes JJ, Bentley WE: Metabolic engineering of the baculovirus expression system via inverse ‘‘shotgun’’ genomic analysis and RNA interference (dsRNA) increases product yield and cell longevity. Biotechnol Bioeng 2007, 98:645-654. 15. Gammell P, Barron N, Kumar N, Clynes M: Initial identification of low temperature and culture stage induction of miRNA expression in suspension CHO-K1 cells. J Biotechnol 2007, 130:213-218. 16. March JC, Bentley WE: Engineering eukaryotic signal transduction with RNAi: enhancing Drosophila S2 cell growth and recombinant protein synthesis via silencing of TSC1. Biotechnol Bioeng 2006, 95:645-652. 17. March JC, Bentley WE: RNAi-based tuning of cell cycling in Drosophila S2 cells — effects on recombinant protein yield. Appl Microbiol Biotechnol 2007, 73:1128-1135. The authors used dsRNA against several genes, including cycE (Cyclin E) and ago (Archipelago) to manipulate the cell cycle in Drosophila S2 cells, increasing the yield of recombinant protein. Furthermore, they demonstrated the importance of ‘tuning’ the level of RNAi suppression, because an intermediate level of cycE silencing resulted in the largest increase in protein production. 18. Hacker DL, Bertschinger M, Baldi L, Wurm FM: Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells. Gene 2004, 341:227-234. 19. Kuystermans D, Krampe B, Swiderek H, Al-Rubeai M: Using cell engineering and omic tools for the improvement of cell culture processes. Cytotechnology 2007, 53:3-22. Overview of current metabolic engineering strategies in cell culture, including RNAi screening and other omic technologies. 20. Mori K, Kuni-Karnochi R, Yarnane-Ohnuki N, Wakitani M, Yamano K, Imai H, Kanda Y, Niwa R, Iida S, Uchida K et al.: Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA. Biotechnol Bioeng 2004, 88:901-908. 21. Kanda Y, Imai-Nishiya H, Kuni-Kamochi R, Mori K, Inoue M, Kitajima-Miyama K, Okazaki A, Iida S, Shitara K, Satoh M: Establishment of a GDP-mannose 4,6-dehydratase (GMD) knockout host cell line: A new strategy for generating Current Opinion in Biotechnology 2008, 19:500–505
504 Tissue, Cell and Pathyway Engineering
completely non-fucosylated recombinant therapeutics. J Biotechnol 2007, 130:300-310. 22. Imai-Nishiya H, Mori K, Inoue M, Wakitani M, Iida S, Shitara K, Satoh M: Double knockdown of alpha 1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in antibodyproducing cells: a new strategy for generating fully nonfucosylated therapeutic antibodies with enhanced ADCC. BMC Biotechnol 2007:7. 23. Ngantung FA, Miller PG, Brushett FR, Tang GL, Wang DIC: RNA interference of sialidase improves glycoprotein sialic acid content consistency. Biotechnol Bioeng 2006, 95:106-119. 24. Lee YY, Wong KTK, Nissom PM, Wong DCF, Yap MGS: Transcriptional profiling of batch and fed-batch protein-free 293-HEK cultures. Metab Eng 2007, 9:52-67. Most recent in a series of papers, along with Wong et al. [11,25] that have used transcriptional profiling to identify metabolic engineering targets during the production cycle of HEK293 (Lee) or CHO (Wong) cells by identifying genes that are upregulated during periods of apoptosis. Potential targets have been successfully suppressed in CHO cells, leading to increased viability and recombinant protein production (Wong). 25. Wong DCF, Wong KTK, Lee YY, Morin PN, Heng CK, Yap M: Transcriptional profiling of apoptotic pathways in batch and fed-batch CHO cell cultures. Biotechnol Bioeng 2006, 94:373-382. See annotation to Ref. [24].
38. Harth G, Zamecnik PC, Tabatadza D, Pierson K, Horwitz MA: Hairpin extensions enhance the efficacy of mycolyl transferase-specific antisense oligonucleotides targeting Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 2007, 104:7199-7204. 39. Meng JR, Hu BQ, Liu J, Hou Z, Meng J, Jia M, Luo XX: Restoration of oxacillin susceptibility in methicillin-resistant Staphylococcus aureus by blocking the MecR1-mediated signaling pathway. J Chemother 2006, 18:360-365. 40. Tan XX, Actor JK, Chen Y: Peptide nucleic acid antisense oligomer as a therapeutic strategy against bacterial infection: proof of principle using mouse intraperitoneal infection. Antimicrob Agents Chemother 2005, 49:3203-3207. 41. Kurupati P, Tan KS, Kumarasinghe G, Pol CJ: Inhibition of gene expression and growth by antisense peptide nucleic acids in a multiresistant beta-lactamase-producing Klebsiella pneummoniae strain. Antimicrob Agents Chemother 2007, 51:805-811. Study demonstrating the potential use of antisense PNAs as a means of preventing infection in antibiotic resistant K. pneumoniae while maintaining the viability of IMR60 epithelial cells. One major issue that remains efficient delivery of PNAs across the cell membrane. 42. Nekhotiaeva N, Awasthi SK, Nielsen PE, Good L: Inhibition of Staphylococcus aureus gene expression and growth using antisense peptide nucleic acids. Mol Ther 2004, 10:625-629.
26. Rasmussen LCV, Sperling-Petersen HU, Mortensen KK: Hitting bacteria at the heart of the central dogma: sequence-specific inhibition. Microb Cell Factories 2007, 6:24. Comprehensive review of antisense technologies in bacteria. Gives more information on fundamental mechanisms, synthesis, and delivery than could be covered in this review.
43. Tilley LD, Hine OS, Kellogg JA, Hassinger JN, Weller DD, Iversen PL, Geller BL: Gene-specific effects of antisense phosphorodiamidate morpholino oligomer–peptide conjugates on Escherichia coli and Salmonella enterica serovar typhimurium in pure culture and in tissue culture. Antimicrob Agents Chemother 2006, 50:2789-2796.
27. Kemmer C, Neubauer P: Antisense RNA based down-regulation of RNaseE in E. coli. Microb Cell Factories 2006, 5:38.
44. Tilley LD, Mellbye BL, Puckett SE, Iversen PL, Geller BL: Antisense peptide-phosphorodiamidate morpholino oligomer conjugate: dose–response in mice infected with Escherichia coli. J Antimicrob Chemother 2007, 59:66-73.
28. Tummala SB, Junne SG, Papoutsakis ET: Antisense RNA downregulation of coenzyme A transferase combined with alcohol–aldehyde dehydrogenase overexpression leads to predominantly alcohologenic Clostridium acetobutylicum fermentations. J Bacteriol 2003, 185:3644-3653. 29. Bouazzaoui K, Lapointe G: Use of antisense RNA to modulate glycosyltransferase gene expression and exopolysaccharide molecular mass in Lactobacillus rhamnosus. J Microb Methods 2006, 65:216-225. 30. Sturino JM, Klaenhammer TR: Engineered bacteriophage defense systems in bioprocessing. Nat Rev Microbiol 2006, 4:395-404. 31. Guerrier-Takada C, Li Y, Altman S: Artificial regulation of gene expression in Escherichia coli by RNase P. Proc Natl Acad Sci U S A 1995, 92:11115-11119. 32. Soler Bistue AJ, Ha H, Sarno R, Don M, Zorreguieta A, Tolmasky ME: External guide sequences targeting the aac(60 )lb mRNA induce inhibition of amikacin resistance. Antimicrob Agents Chemother 2007, 51:1918-1925. 33. McKinney J, Zhang H, Kubori T, Galan JE, Altman S: Disruption of type III secretion in Salmonella enterica serovar Typhimurium by external guide sequences. Nucleic Acids Res 2004, 32:848-854. 34. Sarno R, Ha H, Weinsetel N, Tolmasky ME: Inhibition of aminoglycoside 60 N-acetyltransferase type Ib-mediated amikacin resistance by antisense oligonucleotides. Antimicrob Agents Chemother 2003, 47:3296-3304. 35. Geller BL: Antibacterial antisense. Curr Opin Mol Ther 2005, 7:109-113. 36. Good L: Antisense antibacterials. Expert Opin Ther Patents 2002, 12:1173-1179. 37. Peterson L, de Koning MC, van Kuik-Romeijn P, Weterings J, Pol CJ, Platenburg G, Overhand M, van der Maerl GA, van Boom JH: Synthesis and in vitro evaluation of PNA–peptide– DETA conjugates as potential cell penetrating artificial ribonucleases. Bioconjug Chem 2004, 15:576-582. Current Opinion in Biotechnology 2008, 19:500–505
45. Warfield KL, Panchal RG, Aman MJ, Bavari S: Antisense treatments for biothreat agents. Curr Opin Mol Ther 2006, 8:93-103. 46. Waterhouse PM, Graham MW, Wang MB: Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci U S A 1998, 95:13959-13964. 47. Chuang CF, Meyerowitz EM: Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci U S A 2000, 97:4985-4990. 48. Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM: Total silencing by intron-spliced hairpin RNAs. Nature 2000, 407:319-320. 49. Helliwell CA, Wesley SV, Wielopolska AJ, Waterhouse PM: Highthroughput vectors for efficient gene silencing in plants. Funct Plant Biol 2002, 29:1217-1225. 50. Wielopolska A, Townley H, Moore I, Waterhouse PM, Helliwell CA: A high-throughput inducible RNAi vector for plants. Plant Biotechnol J 2005, 3:583-590. 51. Tang GL, Galili G, Zhuang X: RNAi and microRNA: breakthrough technologies for the improvement of plant nutritional value and metabolic engineering. Metabolomics 2007, 3:357-369. Recent review of RNAi and plant biotechnology. Provides more background, detail, and fundamental information than given in this review. 52. Houmard NM, Mainville JL, Bonin CP, Haung S, Luethy MH, Malvar TM: High-lysine corn generated by endosperm-specific suppression of lysine catabolism using RNAi. Plant Biotechnol J 2007, 5:605-614. 53. Segal G, Song RT, Messing J: A new opaque variant of maize by a single dominant RNA-interference-inducing transgene. Genetics 2003, 165:387-397. 54. Sunilkumar G, Campbell LM, Puckhaber L, Stipanovic RD, Rathore KS: Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proc Natl Acad Sci U S A 2006, 103:18054-18059. www.sciencedirect.com
Beyond silencing— engineering applications of RNA Hebert, Valdes and Bentley 505
The authors used a seed-specific promoter to reduce the toxicity of cottonseed and its oil by suppressing d-cadinene synthase. Expression was not affected in other tissues, maintaining cotton’s natural defense abilities. Although field tests and regulatory hurdles remain, this potentially opens up a novel food source for many people throughout the world. 55. Regina A, Bird A, Topping D, Bowden S, Freeman J, Barsby T, Kosar-Hashemi B, Li Z, Rahman S, Morell M: High-amylose wheat generated by RNAi interference improves indices of large-bowel health in rats. Proc Natl Acad Sci U S A 2006, 103:3546-3551. 56. Dodo HW, Konan KN, Chen FC, Egnin M, Viquez OM: Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant
www.sciencedirect.com
reduction and a decrease in peanut allergenicity. Plant Biotechnol J 2008, 6:135-145. 57. Allen RS, Miller JAC, Chitty JA, Fist AJ, Gerlach WL, Larkin PJ: Metabolic engineering of morphinan alkaloids by overexpression and RNAi suppression of salutaridinol 7-Oacetyltransferase in opium poppy. Plant Biotechnol J 2008, 6:22-30. 58. Qiao F, Yang Q, Wang CL, Fan YL, Wu XF, Zhao KJ: Modification of plant height via RNAi suppression of OsGA20ox2 gene in rice. Euphytica 2007, 158:35-45. 59. Dykxhoorn DM, Lieberman J: The silent revolution: RNA interference as a basic biology, research tool, and therapeutic. Annu Rev Med 2005, 56:401-423.
Current Opinion in Biotechnology 2008, 19:500–505
Beyond silencing — engineering applications of RNA interference and antisense technology for altering cellular phenotype Colin G Hebert1,2,4, James J Valdes2 and William E Bentley1,3,4 Since its discovery 10 years ago, RNA interference (RNAi) has evolved from a research tool into a powerful method for altering the phenotype of cells and whole organisms. Its near universal applicability coupled with its pinpoint accuracy for suppressing target proteins has altered the landscape of many fields. While there is considerable intellectual investment in therapeutics, its potential extends far beyond. In this review, we explore some of these emerging applications — metabolic engineering for enhancing recombinant protein production in both insect and mammalian cell systems, antisense technologies in bacteria as next generation antibiotics, and RNAi in plant biotechnology for improving productivity and nutritional value. Addresses 1 Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant Science Building, College Park, MD 20742, USA 2 U.S. Army Edgewood Chemical Biological Center, AMSRD-ECB-AP-B/ Michel E3330, 5183 Black Hawk RD, Aberdeen Proving Ground, MD 21010, USA 3 Department of Chemical and Biomolecular Engineering, University of Maryland College Park, College Park, MD 20742, USA 4 Room 2330, Jeong H. Kim Engineering Building (Building #225), Fischell Department of Bioengineering, University of Maryland College Park, College Park, MD 20742, USA Corresponding author: Bentley, William E ([email protected])
Current Opinion in Biotechnology 2008, 19:500–505 This review comes from a themed issue on Tissue, Cell and Pathyway Engineering Edited by William Bentley and Michael Betenbaugh
0958-1669/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2008.08.006
Introduction Since its discovery in 1998 [1], RNA interference (RNAi) has quickly moved from a tool for single gene loss-offunction studies to a means of implementing genetic changes in a specific manner over a wide range of organisms and cell types. In addition to unraveling the mechanisms, origins, and purpose of RNAi [2], research has focused on improving and creating new delivery methods for both cells and animals [3], as well as genome-wide RNAi screens for functional analysis [4]. RNAi has also been touted as next great therapeutic tool for combating a wide range of diseases, including cancer, neurodegenerative disease, viral infection, and ocular disorders [5,6]. Great potential exists, but difficulties remain, including Current Opinion in Biotechnology 2008, 19:500–505
effectively delivering the RNAi of interest into the target cell and precisely quantifying specific and nonspecific side effects. Given the numerous reviews and commentary recently published regarding the various aspects of RNAi as an in vivo therapeutic tool [5,6], and the many reviews of the basis for its function, these will not be the focus of this article. Rather, this review seeks to give an overview of some of the applications of RNAi and antisense technology as a means of affecting phenotype at the cellular and organismal level. We describe applications relating to bioprocessing (metabolic engineering for enhancing recombinant protein production) as well as interesting approaches for antisense RNA in bacteria, and, finally, RNAi in plant biotechnology (Figure 1).
RNAi in metabolic engineering Metabolic engineering typically involves the rational alteration of a host cell’s genotype (genes and regulatory structures) in order to achieve a desired phenotype [7,8]. In addition to efficient production of the molecule of interest, desirable phenotypes include the ability to survive well in the adverse conditions, such as decreased oxygen and nutrients or increased cellular and oxidative stress normally experienced by cells overproducing protein [9]. In general, this involves altering the central metabolism, seeking to increase the flux through specific pathways while decreasing others. Although this has traditionally been accomplished through the overexpression of certain proteins, a similar approach can be taken using RNAi and other antisense technologies: instead of overexpressing desirable genes, potentially deleterious genes are silenced. RNAi initially found limited use in metabolic engineering, however, perhaps owing to its transient nature, relatively inefficient delivery methods, and the more typical metabolic engineering objective functions that are based on permanent changes in genotype. As RNAi technology has improved by attempting to address issues such as potency, specificity, and stability, several noteworthy examples based on antisense RNA have appeared targeting apoptosis, the cell growth cycle, and glycosylation. Because apoptosis (programmed cell death) can account for up to 80% of cell death in bioreactors and lead to decreased product yield and quality, antiapoptosis engineering has become an important area of metabolic engineering [10]. This strategy has also been implemented using RNAi. Recent examples include RNAi against proteases in CHO [11,12] and Sf9 cells [13] and www.sciencedirect.com
Beyond silencing— engineering applications of RNA Hebert, Valdes and Bentley 501
Figure 1
RNA interference and altering phenotype. RNA constructs are used in research (screening and loss of function), therapeutics [59], metabolic engineering [16], plant biotechnology, and, in single-stranded form, bacteria.
produced by recombinant baculovirus [14]. In each of these examples, the yield of recombinant protein was enhanced as a result of the downregulation of a target protease. Another approach involves altering the cell growth cycle using RNAi. An optimal production cycle is generally biphasic. The cells first grow quickly to a high density before transitioning to a production phase characterized by low growth and high productivity [15]. Achieving this regime is relatively straightforward in bacterial systems, but can be more problematic in eukaryotic cell culture because their state is influenced by the cell cycle. Although past efforts have used more traditional metabolic engineering methods for controlling the cell cycle, recent studies have used RNAi to increase cell growth and protein production in Drosophila S2 cells through the downregulation of cell growth controllers [16,17] and in HEK 293 cells through the downregulation of transcriptional regulators [18]. A final area of metabolic engineering in which RNAi has played a role is glycosylation. Engineering specific glycosylation patterns can increase the potency and stability of therapeutic proteins while at the same time decreasing their immunogenic effects [19]. Several specific examples of using RNAi to fine-tune glycosylation patterns include increasing the antibody-dependent cellular cytotoxicity of antibodies produced in CHO cells through the reduction of a 1,6-fucosyltransferase (FUT8) [20], GDP mannose-4,6-dehydrogenase (GMP) [21], or both FUT8 and GMP [22], as well as improving product quality by reducing sialidase activity in CHO cells [23]. www.sciencedirect.com
As the amount of genomic and metabolomic data increases, additional RNAi-based metabolic engineering targets will surely be identified. Some such screens are already underway [15,24,25], and as more targets are identified, RNAi will continue to expand its influence in the field of metabolic engineering.
Antisense RNA in prokaryotic systems Although the majority of RNA-based cellular engineering has focused on eukaryotic systems, antisense RNA technology has also been employed in bacterial systems, both as a metabolic engineering tool and a potential therapeutic agent. Antisense molecules anneal to complementary mRNA strands, either by blocking translation through steric hindrance or causing rapid degradation, potentially by dsRNA-specific RNases. The efficiency of this disruption depends upon several factors, including the length and structure of the antisense molecule, as well as the intracellular concentration and resistance to the degradation of the target mRNA [26]. In addition to the loss-of-function studies performed in many organisms [26], asRNA has been used as a metabolic engineering tool to enhance the productivity of several bacterial hosts including E. coli [27], Clostridium acetobutylicum [28], and Lactobacillus rhamnosus [29]. Antisense approaches have also been used to protect bacteria against bacteriophages [30]. Another application of asRNA as a means of altering bacterial phenotype is through a reduction in pathogenicity, either by reducing the potency of certain toxins or virulence mechanisms, or through the recovery of antibiotic susceptibility. External guide sequences (EGS), which are small asRNAs expressed from plasmids, bind Current Opinion in Biotechnology 2008, 19:500–505
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complementary mRNA to activate cleavage through the endogenous RNase P activity and have also been used to alter bacterial phenotype. Initial studies used EGS to reduce the levels of alkaline phosphatase and b-galactosidase [31], but further applications in E. coli have restored antibiotic sensitivity in E. coli [32] and decreased host cell invasion rate in Salmonella enterica serovar Typhimurium [33]. Although EGS have been successful in a laboratory setting, their potential as an in vivo therapeutic is somewhat limited because they are generally more effective when expressed on plasmids. As an alternative, many researchers have turned to antisense phosphodiester oligodeoxyribonucleotides (asODNs), which are short, chemically synthesized DNA sequences complementary to the mRNA of the target gene. ODNs have been used to decrease antibiotic resistance in E. coli [34], but have seen limited use directly owing to their rapid degradation by nucleases. Rather, a number of synthetic analogs have been developed, the most promising of which include phosphorothiate ODNs (PS-ODNs), peptide nucleic acids (PNAs), and phosphorodiamidate morpholino oligomers (PMOs) [35–37]. PS-ODNs have been well studied in eukaryotic systems, but have so far seen limited use in bacterial systems, mostly in Mycobacteria. Recent examples include the application of PS-ODNs to inhibit growth in M. tuberculosis [38] and restore antibiotic susceptibility in a methicillin-resistant strain of S. aureus [39]. PNAs have been used to target a number of RNAs in E. coli [40], the Gramnegative human pathogen Klebsiella pneumoniae [41], and S. aureus [42], resulting in inhibited growth, though sometimes at relatively high concentrations. The study involving K. pneumoniae used PNAs targeting two essential genes to cure infected IMR90 cells in culture [41]. Applications involving PMOs have mainly targeted the acyl carrier protein of E. coli [43,44], though efforts are underway to test their ability to silence genes in Bacillus anthrax as well [45]. Despite the success of these synthetic analogs, many challenges remain before they can be successfully applied as antimicrobials, including solubility, binding efficiency, resistance to degradation, and cellular delivery, which is perhaps the most pressing [26]. Antisense technologies have shown great effectiveness in in vitro and cell-free systems; however, they will not emerge as next generation antimicrobial drugs until they can be effectively delivered to their target. As bacteria become increasingly resistant to the current methods of treatment, so increases the importance of developing new antisense technologies.
and antisense RNA against the same gene, dsRNA technology was not applied in plants until 2000, when flower identity genes were silenced used inverted repeats [47]. RNAi technology in plants has been successfully implemented using both intron spliced long dsRNA and hairpin RNAs to silence target genes [48,49]. Chemically inducible vectors have also been developed [50], but are not as practical or safe for large-scale use as tissue-specific or organ-specific promoters for the development of RNAi technology in plants [51]. The main thrust of RNAi application in plants is for the improvement of plant productivity or nutritional value [51]. Several groups have increased the lysine content in corn either through the knockdown of lysine–ketoglutarate reductase/saccharophine dehydrogenase (ZLRK/ SDH), the enzyme responsible for its catabolism [52], or the suppression of specific zein storage proteins [53]. Through the use of an endosperm-specific promoter, Houmard et al. confined the increase in lysine content to the kernels, reducing side effects such as abnormal growth and flower development that can be present in plants overproducing lysine in all tissues [52]. Because lysine is one of the essential amino acids, its introduction into corn has worldwide implications in many western countries where it is used mainly as a livestock feed (and is currently supplemented with lysine) as well as in developing countries, where corn is staple crop for humans. Another application of RNAi to an agriculturally important crop is the development of cottonseed with a reduced amount of gossypol toxin [54]. Through the use of a seed-specific promoter, Sunilkumar et al. were able to reduce the amount of gossypol in cottonseeds well below that deemed safe for human consumption by United Nations Food and Agriculture Organization and World Health Organization standards, potentially opening up a new food source for millions of people in the developing world, while maintaining wild-type levels in leaves and other tissues, preserving their function as protection against insects and disease [54]. This approach in cottonseed has laid the groundwork for the development of similar approaches in other potential food sources that can be hampered by their toxicity, including the tropical legume Lathyrus sativus, cassava, and fava beans [51]. Other examples of RNAi technology for the improvement of plant nutritional value include the development of high-fiber wheat [55], as well as a reduction in the allergenicity of peanuts [56]. Two final examples include modifying the profile of morphinan alkaloids in opium poppy [57], as well as modifying rice plant height to increase lodging resistance and decrease potential damage from wind and rain [58].
RNAi in plants Despite the discovery of post-transcriptional gene silencing in 1998 [46] by crossing plants that produced sense Current Opinion in Biotechnology 2008, 19:500–505
The application of RNAi with the potential to impact the greatest number of people may be in plant biotechnology. www.sciencedirect.com
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In many developing countries, people depend upon certain plants not only for income, but also as a means of survival. As global populations and demand for food continue to increase, new agricultural technologies and techniques will be required to meet that demand in a responsible and sustainable manner. Current strategies include improving the productivity or nutritional value of existing food crops or developing new ones. RNAi has emerged as an important tool in both these areas.
Conclusion Although RNAi has received the most attention for its potential as a next generation therapeutic, the alternate applications presented here are no less important. Metabolic engineering via both RNAi and antisense RNA impact the production of not only therapeutic proteins and antibodies, but also biologically based fuels and chemicals, which is a field that will become increasingly important as resources become limited and sustainability becomes a higher priority. Antisense molecules targeting antibiotic resistant and other pathogenic bacteria will also become important as more bacteria evolve resistance to current antibiotic treatments. Finally, the enhancement of plants through the use of RNAi, either as a means of improving current food crops, such as corn and rice, or developing new ones, such as cottonseed, has the potential to impact the lives of millions of people throughout the world. As the fundamental knowledge and associated technology associated with RNAi continues to increase, applications already under study will improve. This is perhaps one of the marks of a great discovery, that its influence has extended well beyond the use for which it was originally intended. It will be interesting to see what additional applications are developed as RNAi enters into its second decade.
Acknowledgements Partial support of this work was provided by the Department of Defense (CREST Fellowship to CGH) and the National Institutes of Health (GM70851-01).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC: Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998, 391:806-811.
2.
Tomari Y, Zamore PD: Perspective: machines for RNAi. Genes Dev 2005, 19:517-529.
3.
Kumar LD, Clarke AR: Gene manipulation through the use of small interfering RNA (siRNA): from in vitro to in vivo applications. Adv Drug Deliv Rev 2007, 59:87-100.
4.
Echeverri CJ: High-throughput RNAi screening in cultured cells: a user’s guide. Nat Rev Genet 2006, 7:373-383.
5.
de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J: Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov 2007, 6:443-453.
www.sciencedirect.com
6.
Dykxhoorn DM, Palliser D, Lieberman J: The silent treatment: siRNAs as small molecule drugs. Gene Therapy 2006, 13:541-552. Recent and comprehensive review of RNAi as a therapeutic. Discusses fundamentals, delivery technologies, and siRNA efficacy against specific diseases. 7.
Bailey JE: Toward a science of metabolic engineering. Science 1991, 252:1668-1675.
8.
Stephanopoulos G, Vallino JJ: Network rigidity and metabolic engineering in metabolite overproduction. Science 1991, 252:1675-1681.
9.
Rao RV, Bredesen DE: Misfolded proteins, endoplasmic reticulum stress and neurodegeneration. Curr Opin Cell Biol 2004, 16:653-663.
10. Majors BS, Betenbaugh MJ, Chiang GG: Links between metabolism and apoptosis in mammalian cells: applications for anti-apoptosis engineering. Metab Eng 2007, 9:317-326. 11. Wong DCF, Wong KTK, Nissom PM, Heng CK, Yap MGS: Targeting early apoptotic genes in batch and fed-batch CHO cell cultures. Biotechnol Bioeng 2006, 95:350-361. See annotation to Ref. [24]. 12. Lai DZ, Fu L, Weng SJ, Qi LQ, Yu CM, Yu T, Wang HT, Chen W: Blocking caspase-3 activity with a U6SnRNA promoter-driven ribozyme enhances survivability of CHO cells cultured in low serum medium and production of interferon-beta. Biotechnol Bioeng 2004, 85:20-28. 13. Lin CC, Hsu JTA, Huang KL, Tang HK, Shu CW, Lai YK: SfCaspase-I-repressed stable cells: resistance to apoptosis and augmentation of recombinant protein production. Biotechnol Appl Biochem 2007, 48:11-19. 14. Kim EJ, Kramer SF, Hebert CG, Valdes JJ, Bentley WE: Metabolic engineering of the baculovirus expression system via inverse ‘‘shotgun’’ genomic analysis and RNA interference (dsRNA) increases product yield and cell longevity. Biotechnol Bioeng 2007, 98:645-654. 15. Gammell P, Barron N, Kumar N, Clynes M: Initial identification of low temperature and culture stage induction of miRNA expression in suspension CHO-K1 cells. J Biotechnol 2007, 130:213-218. 16. March JC, Bentley WE: Engineering eukaryotic signal transduction with RNAi: enhancing Drosophila S2 cell growth and recombinant protein synthesis via silencing of TSC1. Biotechnol Bioeng 2006, 95:645-652. 17. March JC, Bentley WE: RNAi-based tuning of cell cycling in Drosophila S2 cells — effects on recombinant protein yield. Appl Microbiol Biotechnol 2007, 73:1128-1135. The authors used dsRNA against several genes, including cycE (Cyclin E) and ago (Archipelago) to manipulate the cell cycle in Drosophila S2 cells, increasing the yield of recombinant protein. Furthermore, they demonstrated the importance of ‘tuning’ the level of RNAi suppression, because an intermediate level of cycE silencing resulted in the largest increase in protein production. 18. Hacker DL, Bertschinger M, Baldi L, Wurm FM: Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells. Gene 2004, 341:227-234. 19. Kuystermans D, Krampe B, Swiderek H, Al-Rubeai M: Using cell engineering and omic tools for the improvement of cell culture processes. Cytotechnology 2007, 53:3-22. Overview of current metabolic engineering strategies in cell culture, including RNAi screening and other omic technologies. 20. Mori K, Kuni-Karnochi R, Yarnane-Ohnuki N, Wakitani M, Yamano K, Imai H, Kanda Y, Niwa R, Iida S, Uchida K et al.: Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA. Biotechnol Bioeng 2004, 88:901-908. 21. Kanda Y, Imai-Nishiya H, Kuni-Kamochi R, Mori K, Inoue M, Kitajima-Miyama K, Okazaki A, Iida S, Shitara K, Satoh M: Establishment of a GDP-mannose 4,6-dehydratase (GMD) knockout host cell line: A new strategy for generating Current Opinion in Biotechnology 2008, 19:500–505
504 Tissue, Cell and Pathyway Engineering
completely non-fucosylated recombinant therapeutics. J Biotechnol 2007, 130:300-310. 22. Imai-Nishiya H, Mori K, Inoue M, Wakitani M, Iida S, Shitara K, Satoh M: Double knockdown of alpha 1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in antibodyproducing cells: a new strategy for generating fully nonfucosylated therapeutic antibodies with enhanced ADCC. BMC Biotechnol 2007:7. 23. Ngantung FA, Miller PG, Brushett FR, Tang GL, Wang DIC: RNA interference of sialidase improves glycoprotein sialic acid content consistency. Biotechnol Bioeng 2006, 95:106-119. 24. Lee YY, Wong KTK, Nissom PM, Wong DCF, Yap MGS: Transcriptional profiling of batch and fed-batch protein-free 293-HEK cultures. Metab Eng 2007, 9:52-67. Most recent in a series of papers, along with Wong et al. [11,25] that have used transcriptional profiling to identify metabolic engineering targets during the production cycle of HEK293 (Lee) or CHO (Wong) cells by identifying genes that are upregulated during periods of apoptosis. Potential targets have been successfully suppressed in CHO cells, leading to increased viability and recombinant protein production (Wong). 25. Wong DCF, Wong KTK, Lee YY, Morin PN, Heng CK, Yap M: Transcriptional profiling of apoptotic pathways in batch and fed-batch CHO cell cultures. Biotechnol Bioeng 2006, 94:373-382. See annotation to Ref. [24].
38. Harth G, Zamecnik PC, Tabatadza D, Pierson K, Horwitz MA: Hairpin extensions enhance the efficacy of mycolyl transferase-specific antisense oligonucleotides targeting Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 2007, 104:7199-7204. 39. Meng JR, Hu BQ, Liu J, Hou Z, Meng J, Jia M, Luo XX: Restoration of oxacillin susceptibility in methicillin-resistant Staphylococcus aureus by blocking the MecR1-mediated signaling pathway. J Chemother 2006, 18:360-365. 40. Tan XX, Actor JK, Chen Y: Peptide nucleic acid antisense oligomer as a therapeutic strategy against bacterial infection: proof of principle using mouse intraperitoneal infection. Antimicrob Agents Chemother 2005, 49:3203-3207. 41. Kurupati P, Tan KS, Kumarasinghe G, Pol CJ: Inhibition of gene expression and growth by antisense peptide nucleic acids in a multiresistant beta-lactamase-producing Klebsiella pneummoniae strain. Antimicrob Agents Chemother 2007, 51:805-811. Study demonstrating the potential use of antisense PNAs as a means of preventing infection in antibiotic resistant K. pneumoniae while maintaining the viability of IMR60 epithelial cells. One major issue that remains efficient delivery of PNAs across the cell membrane. 42. Nekhotiaeva N, Awasthi SK, Nielsen PE, Good L: Inhibition of Staphylococcus aureus gene expression and growth using antisense peptide nucleic acids. Mol Ther 2004, 10:625-629.
26. Rasmussen LCV, Sperling-Petersen HU, Mortensen KK: Hitting bacteria at the heart of the central dogma: sequence-specific inhibition. Microb Cell Factories 2007, 6:24. Comprehensive review of antisense technologies in bacteria. Gives more information on fundamental mechanisms, synthesis, and delivery than could be covered in this review.
43. Tilley LD, Hine OS, Kellogg JA, Hassinger JN, Weller DD, Iversen PL, Geller BL: Gene-specific effects of antisense phosphorodiamidate morpholino oligomer–peptide conjugates on Escherichia coli and Salmonella enterica serovar typhimurium in pure culture and in tissue culture. Antimicrob Agents Chemother 2006, 50:2789-2796.
27. Kemmer C, Neubauer P: Antisense RNA based down-regulation of RNaseE in E. coli. Microb Cell Factories 2006, 5:38.
44. Tilley LD, Mellbye BL, Puckett SE, Iversen PL, Geller BL: Antisense peptide-phosphorodiamidate morpholino oligomer conjugate: dose–response in mice infected with Escherichia coli. J Antimicrob Chemother 2007, 59:66-73.
28. Tummala SB, Junne SG, Papoutsakis ET: Antisense RNA downregulation of coenzyme A transferase combined with alcohol–aldehyde dehydrogenase overexpression leads to predominantly alcohologenic Clostridium acetobutylicum fermentations. J Bacteriol 2003, 185:3644-3653. 29. Bouazzaoui K, Lapointe G: Use of antisense RNA to modulate glycosyltransferase gene expression and exopolysaccharide molecular mass in Lactobacillus rhamnosus. J Microb Methods 2006, 65:216-225. 30. Sturino JM, Klaenhammer TR: Engineered bacteriophage defense systems in bioprocessing. Nat Rev Microbiol 2006, 4:395-404. 31. Guerrier-Takada C, Li Y, Altman S: Artificial regulation of gene expression in Escherichia coli by RNase P. Proc Natl Acad Sci U S A 1995, 92:11115-11119. 32. Soler Bistue AJ, Ha H, Sarno R, Don M, Zorreguieta A, Tolmasky ME: External guide sequences targeting the aac(60 )lb mRNA induce inhibition of amikacin resistance. Antimicrob Agents Chemother 2007, 51:1918-1925. 33. McKinney J, Zhang H, Kubori T, Galan JE, Altman S: Disruption of type III secretion in Salmonella enterica serovar Typhimurium by external guide sequences. Nucleic Acids Res 2004, 32:848-854. 34. Sarno R, Ha H, Weinsetel N, Tolmasky ME: Inhibition of aminoglycoside 60 N-acetyltransferase type Ib-mediated amikacin resistance by antisense oligonucleotides. Antimicrob Agents Chemother 2003, 47:3296-3304. 35. Geller BL: Antibacterial antisense. Curr Opin Mol Ther 2005, 7:109-113. 36. Good L: Antisense antibacterials. Expert Opin Ther Patents 2002, 12:1173-1179. 37. Peterson L, de Koning MC, van Kuik-Romeijn P, Weterings J, Pol CJ, Platenburg G, Overhand M, van der Maerl GA, van Boom JH: Synthesis and in vitro evaluation of PNA–peptide– DETA conjugates as potential cell penetrating artificial ribonucleases. Bioconjug Chem 2004, 15:576-582. Current Opinion in Biotechnology 2008, 19:500–505
45. Warfield KL, Panchal RG, Aman MJ, Bavari S: Antisense treatments for biothreat agents. Curr Opin Mol Ther 2006, 8:93-103. 46. Waterhouse PM, Graham MW, Wang MB: Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci U S A 1998, 95:13959-13964. 47. Chuang CF, Meyerowitz EM: Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci U S A 2000, 97:4985-4990. 48. Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM: Total silencing by intron-spliced hairpin RNAs. Nature 2000, 407:319-320. 49. Helliwell CA, Wesley SV, Wielopolska AJ, Waterhouse PM: Highthroughput vectors for efficient gene silencing in plants. Funct Plant Biol 2002, 29:1217-1225. 50. Wielopolska A, Townley H, Moore I, Waterhouse PM, Helliwell CA: A high-throughput inducible RNAi vector for plants. Plant Biotechnol J 2005, 3:583-590. 51. Tang GL, Galili G, Zhuang X: RNAi and microRNA: breakthrough technologies for the improvement of plant nutritional value and metabolic engineering. Metabolomics 2007, 3:357-369. Recent review of RNAi and plant biotechnology. Provides more background, detail, and fundamental information than given in this review. 52. Houmard NM, Mainville JL, Bonin CP, Haung S, Luethy MH, Malvar TM: High-lysine corn generated by endosperm-specific suppression of lysine catabolism using RNAi. Plant Biotechnol J 2007, 5:605-614. 53. Segal G, Song RT, Messing J: A new opaque variant of maize by a single dominant RNA-interference-inducing transgene. Genetics 2003, 165:387-397. 54. Sunilkumar G, Campbell LM, Puckhaber L, Stipanovic RD, Rathore KS: Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proc Natl Acad Sci U S A 2006, 103:18054-18059. www.sciencedirect.com
Beyond silencing— engineering applications of RNA Hebert, Valdes and Bentley 505
The authors used a seed-specific promoter to reduce the toxicity of cottonseed and its oil by suppressing d-cadinene synthase. Expression was not affected in other tissues, maintaining cotton’s natural defense abilities. Although field tests and regulatory hurdles remain, this potentially opens up a novel food source for many people throughout the world. 55. Regina A, Bird A, Topping D, Bowden S, Freeman J, Barsby T, Kosar-Hashemi B, Li Z, Rahman S, Morell M: High-amylose wheat generated by RNAi interference improves indices of large-bowel health in rats. Proc Natl Acad Sci U S A 2006, 103:3546-3551. 56. Dodo HW, Konan KN, Chen FC, Egnin M, Viquez OM: Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant
www.sciencedirect.com
reduction and a decrease in peanut allergenicity. Plant Biotechnol J 2008, 6:135-145. 57. Allen RS, Miller JAC, Chitty JA, Fist AJ, Gerlach WL, Larkin PJ: Metabolic engineering of morphinan alkaloids by overexpression and RNAi suppression of salutaridinol 7-Oacetyltransferase in opium poppy. Plant Biotechnol J 2008, 6:22-30. 58. Qiao F, Yang Q, Wang CL, Fan YL, Wu XF, Zhao KJ: Modification of plant height via RNAi suppression of OsGA20ox2 gene in rice. Euphytica 2007, 158:35-45. 59. Dykxhoorn DM, Lieberman J: The silent revolution: RNA interference as a basic biology, research tool, and therapeutic. Annu Rev Med 2005, 56:401-423.
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