Choosing siRNA, shRNA, and miRNA for Gene Silencing

RNAi refers to dsRNA-induced gene silencing, a cellular process that degrades RNA homologous to one strand of the dsRNA [1, 2]. The intermediates of long dsRNA-initiated RNAi are double-stranded small interfering RNAs (siRNA), typically 21-23 nucleotide (nt) long. The siRNAs, when introduced into cells, can be used to silence genes in mammalian systems where long dsRNAs prompt protein kinase R (PKR), RNase L, and interferon activities that result in non-specific RNA degradation and general shutdown of protein synthesis [3]. siRNAs can either be chemically synthesized then directly transfected into cells or can be generated inside the cell by introducing vectors that express short-hairpin RNA (shRNA) precursors of siRNAs. The process of shRNA into functional siRNA involves cellular RNAi machinery that naturally process genome encoded microRNAs (miRNA) that are responsible for cellular regulation of gene expression by modulating mRNA stability, translation, and chromatin structures [4].

Chemically synthesized siRNA is the simplest format for RNAi. One of the biggest hurdles for achieving effective RNAi with siRNA is that many cells are difficult to transfect. An RNAi experiment is typically considered successful when the target gene expression is reduced by >70%, a threshold not reachable by many types of cells due to their low transfection efficiency. Another drawback of using synthetic siRNA is the limited duration of post-transfection effects, typically with gene silencing activities peaking around 24 hours, and diminishing within 48 hours [5]. Chemical synthesis of siRNA, which is a service Allele Biotech and Orbigen (now merged under the Allele brand) pioneered and still provides, is expensive on a per transfection basis relative to DNA vector based reagents.

shRNA can be introduced by DNA plasmid, linear template, or packaged retroviral/lentiviral vectors. Using any form of DNA construct, except the PCR template format such as Allele’s LineSilence platform, requires creating DNA constructs and sequence verification; a taxing work load if multiple genes need to be studied. However, once the constructs are made, they can be reproduced easily and inexpensively. It is difficult to directly compare the effectiveness of siRNA versus shRNA on a per molecule basis because RNA polymerase III (Pol III) promoters such as U6 or H1 commonly used to express shRNAs can make thousands of copies of shRNA from a single DNA template. However when both siRNA and shRNA are produced the same way, e.g. synthesized chemically, shRNA is reported to be somewhat more effective [6, 7]. For the goals of this research, the most important advantage using shRNA can provide over siRNA is that it can be carried on a lentiviral vector and introduced into a wide variety of cells.

Similar to the comparison between siRNA versus shRNA, it is also difficult to rank the efficiency of shRNA versus miRNA from published data, partly due to different results from different experimental systems. There have been several reports that showed shRNA can cause significant cell toxicity, especially in vivo such as after injection into mouse brain. It was originally reasoned that highly efficient expression from Pol III promoters might overwhelm the cellular machinery that is needed to execute endogenous RNAi functions such as transporting miRNA from the nucleus to the cytoplasm. It was later found out that even using Pol III promoter to create miRNA could still mitigate the toxic effects of shRNA [8]. Since shRNA and miRNA are processed by endonuclease Dicer before being incorporated into RNA induced silencing complex (RISC), the exact identity of siRNAs produced from a given shRNA or miRNA targeting the same region on the mRNA are not known in most of the earlier studies. By designing shRNA and miRNA to give exactly the same processed siRNAs, Boudreau et al. showed that shRNA is actually more potent than miRNA in various systems [9].

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1. Fire, A., S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, and C.C. Mello, Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998. 391(6669): p. 806-11.
2. Hannon, G.J., RNA interference. Nature, 2002. 418(6894): p. 244-51.
3. McManus, M.T. and P.A. Sharp, Gene silencing in mammals by small interfering RNAs. Nat Rev Genet, 2002. 3(10): p. 737-47.
4. Hutvagner, G. and P.D. Zamore, A microRNA in a multiple-turnover RNAi enzyme complex. Science, 2002. 297(5589): p. 2056-60.
5. Rao, D.D., J.S. Vorhies, N. Senzer, and J. Nemunaitis, siRNA vs. shRNA: similarities and differences. Adv Drug Deliv Rev, 2009. 61(9): p. 746-59.
6. Vlassov, A.V., B. Korba, K. Farrar, S. Mukerjee, A.A. Seyhan, H. Ilves, R.L. Kaspar, D. Leake, S.A. Kazakov, and B.H. Johnston, shRNAs targeting hepatitis C: effects of sequence and structural features, and comparision with siRNA. Oligonucleotides, 2007. 17(2): p. 223-36.
7. Siolas, D., C. Lerner, J. Burchard, W. Ge, P.S. Linsley, P.J. Paddison, G.J. Hannon, and M.A. Cleary, Synthetic shRNAs as potent RNAi triggers. Nat Biotechnol, 2005. 23(2): p. 227-31.
8. McBride, J.L., R.L. Boudreau, S.Q. Harper, P.D. Staber, A.M. Monteys, I. Martins, B.L. Gilmore, H. Burstein, R.W. Peluso, B. Polisky, B.J. Carter, and B.L. Davidson, Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi. Proc Natl Acad Sci U S A, 2008. 105(15): p. 5868-73.
9. Boudreau, R.L., A.M. Monteys, and B.L. Davidson, Minimizing variables among hairpin-based RNAi vectors reveals the potency of shRNAs. Rna, 2008. 14(9): p. 1834-44.

Tags: alpaca antibodies, camelid antibody, gene knockdown, gene silencing, GFP-Trap, lentivirus packaging, miRNA, nanobodies, packaging systems, RFP-Trap, RNAi, shRNA, shRNA packaging, siRNA, VHH, virus packaging service

What seems to be going on with RNAi related patents in the US

Reciting Table 1 from Ref 1 and Table 3 from Ref 2:

By the end Nov 2008 it appears that Allele’s patent (US 7,294,504 and 7,422,896) are the only currently granted DNA based RNAi patents. The focus of Allele’s technology is siRNA of 21-23, either in separate sense and antisense strands, or shRNA or miRNA format, thus not covered by the Fire patent or the Kreutzer-Limmer patent. Since these RNAi inducers are not synthesized by chemical reactions, or produced with enzymes or cell lysate in vitro, they do not relate to Tuschl I or II patent groups. Allele Biotech can not guarantee that its interpretation is correct or final by any means; commercial user of any of the related technologies should perform own due diligence.

[1] Charlie Schmidt. March 2007 “Negotiating the RNAi patent thicket” Nature Biotechnology 25 (3): 273-275

[2] Dirk Haussecker. May 2008 “The Business of RNAi Therapeutics” Human Gene Therapy 19: 451-462

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Tags: gene silencing, H1, lentiviral vector, miRNA, PCR cassette, plasmid vector, pol III, retroviral vector, RNAi, shRNA, siRNA, U6