Skip to main content
SpringerLink
Log in

RNA Interference: Mechanisms, Technical Challenges, and Therapeutic Opportunities

  • Protocol
  • First Online:
RNA Interference

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1218))

Abstract

The ability to inhibit gene expression via RNA interference (RNAi) has a broad therapeutic potential for various human diseases such as infections and cancers. Recent advances in mechanistic understanding of RNAi have improved the design of functional small interfering (si) RNAs with superior potency and specificity. With respect to delivery, new developments in delivery strategies have facilitated preclinical and clinical siRNA applications. This review provides valuable insights to guide the design and delivery of therapeutic siRNAs.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811

    Article  PubMed  CAS  Google Scholar 

  2. Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950–952

    Article  PubMed  CAS  Google Scholar 

  3. Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25–33

    Article  PubMed  CAS  Google Scholar 

  4. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498

    Article  PubMed  CAS  Google Scholar 

  5. Yan KS, Yan S, Farooq A, Han A, Zeng L, Zhou MM (2003) Structure and conserved RNA binding of the PAZ domain. Nature 426:468–474

    Article  PubMed  Google Scholar 

  6. Pencheva N, Tavazoie SF (2013) Control of metastatic progression by microRNA regulatory networks. Nat Cell Biol 15:546–554

    Article  PubMed  CAS  Google Scholar 

  7. Zamore PD, Haley B (2005) Ribogenome: the big word of small RNAs. Science 309:1519–1524

    Article  PubMed  CAS  Google Scholar 

  8. Kim VN (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 6:376–385

    Article  PubMed  CAS  Google Scholar 

  9. Liu Q, Rand TA, Kalidas S, Du F, Kim HE, Smith DP, Wang X (2003) R2d2, a bridge between the initiation and effector steps of the drosophila RNAi pathway. Science 301:1921–1925

    Article  PubMed  CAS  Google Scholar 

  10. Liu X, Jiang F, Kalidas S, Smith D, Liu Q (2006) Dicer-2 and R2D2 coordinately bind siRNA to promote assembly of the siRISC complexes. RNA 12:1514–1520

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  11. Matranga C, Tomari Y, Shin C, Bartel DP, Zamore PD (2005) Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123:607–620

    Article  PubMed  CAS  Google Scholar 

  12. Wee LM, Flores-Jasso CF, Salomon WE, Zamore PD (2012) Argonaute divides its RNA guide into domains with distinct functions and RNA-binding properties. Cell 151:1055–1067

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  13. Holen T, Amarzguiouni M, Wiiger MT, Babaie E, Prydz H (2002) Positional effects of short interfering RNAs targeting the human coagulation trigger tissue factor. Nucleic Acids Res 30:1757–1766

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  14. Tomari Y, Matranga C, Haley B, Martinez N, Zamore PD (2004) A protein sensor for siRNA asymmetry. Science 306:1377–1380

    Article  PubMed  CAS  Google Scholar 

  15. Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N et al (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115:199–208

    Article  PubMed  CAS  Google Scholar 

  16. Reyolds A, Leake D, Boese Q, Scaringe S, Marchall WS et al (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22:326–330

    Article  Google Scholar 

  17. Ui-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H et al (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res 32:936–948

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  18. Sano M, Sierant M, Miyagishi M, Nakanishi M, Takagi Y, Sutou S (2008) Effect of asymmetric terminal structures of short RNA duplexes on the RNA interference activity and strand selection. Nucleic Acids Res 36:5812–5821

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  19. Jackson AL, Burchard J, Leake D, Reynolds A, Schelter J, Guo J, Johnson JM, Lim L, Karpilow J, Nichols K, Marshall W, Khvorova A, Linsley PS (2006) Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA 12:1197–1205

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  20. Paul CP, Good PD, Winer I et al (2002) Effective expression of small interference RNA in human cells. Nat Biotechnol 20:505–508

    Article  PubMed  CAS  Google Scholar 

  21. Watts JK, Deleavey GF, Damha MJ (2008) Chemically modified siRNA: tools and applications. Drug Discov Today 13:842–855

    Article  PubMed  CAS  Google Scholar 

  22. Hoerter JA, Walter NG (2007) Chemical modification resolves the asymmetry of siRNA strand degradation in human blood serum. RNA 13:1887–1893

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  23. Mook OR, Baas F, de Wissel MB, Fluiter K (2007) Evaluation of locked nucleic acid-modified small interfering RNA in vitro and in vivo. Mol Cancer Ther 6:833–843

    Article  PubMed  CAS  Google Scholar 

  24. Laursen MB, Pakula MM, Gao S, Fluiter K, Mook OR, Baas F et al (2010) Utilisation of unlocked nucleic acid to enhance siRNA performance in vitro and in vivo. Mol Biosyst 6:862–870

    Article  PubMed  CAS  Google Scholar 

  25. Sioud M (2006) Single-stranded small interfering RNA are more immunostimulatory than their double-stranded counterparts: a central role for 2′-hydroxyl uridines in immune responses. Eur J Immunol 36:1222–1230

    Article  PubMed  CAS  Google Scholar 

  26. Rettig GR, Behlke MA (2012) Progress toward in vivo use of siRNAs-ii. Mol Ther 20:483–512

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  27. Tammali R, Reddy AB, Saxena A, Rychahou PG, Evers BM, Qiu S, Awasthi S, Ramana KV, Srivastava SK (2011) Inhibition of aldose reductase prevents colon cancer metastasis. Carcinogenesis 32:1259–1267

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  28. Huang Y et al (2011) Elimination pathways of systemically delivered siRNA. Mol Ther 19:381–385

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  29. Barton GM, Medzhitov R (2002) Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci U S A 99:14943–14945

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  30. Winston WM, Molodowitch C, Hunter CP (2002) Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. Science 295:2456–2459

    Article  PubMed  CAS  Google Scholar 

  31. Saleh MC, Van Rij RP, Hekele A, Gillis A, Foley E, O’Farrell PH, Andino R (2006) The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing. Nat Cell Biol 8:793–802

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  32. Feinberg EH, Hunter CP (2003) Transport of dsRNA into cells by the transmembrane protein SID-1. Science 301:1545–1547

    Article  PubMed  CAS  Google Scholar 

  33. Fougerolles AD, Vornlocher HP, Maraganore J et al (2007) Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov 6:443–453

    Article  PubMed  Google Scholar 

  34. Kanastry R, Dorkin JR, Vegas A, Anderson D (2013) Delivery siRNA therapeutics. Nat Mater 12:967–977

    Article  Google Scholar 

  35. Simoes S, Filipe A, Faneca H, Mano M, Penacho N, Duzgunes N, De Lima MP (2005) Cationic liposomes for gene delivery. Expert Opin Drug Deliv 2:237–254

    Article  PubMed  CAS  Google Scholar 

  36. Sun TM, Du JZ, Yan LF et al (2008) Self-assembled biodegradable micellar nanoparticles of amphiphilic and cationic block copolymer for siRNA delivery. Biomaterials 29:4348–4355

    Article  PubMed  CAS  Google Scholar 

  37. Peer D, Karp JM, Hong S et al (2007) Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol 2:751–760

    Article  PubMed  CAS  Google Scholar 

  38. Gomes-Da-Silva LC, Fonseca NA, Moura V, Pedroso De Lima MC, Simoes S, Moreira JN (2012) Lipid-based nanoparticles for siRNA delivery in cancer therapy: paradigms and challenges. Acc Chem Res 45:1163–1171

    Article  PubMed  CAS  Google Scholar 

  39. Morrissey DV, Lockridge JA, 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 CS, Jeffs LB, Judge A, Maclachlan I, Polisky B (2005) Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat Biotechnol 23:1002–1007

    Article  PubMed  CAS  Google Scholar 

  40. Zimmermann TS, Lee AC, Akinc A, Bramlage B, Bumcrot D, Fedoruk MN et al (2006) RNAi-mediated gene silencing in non-human primates. Nature 441:111–114

    Article  PubMed  CAS  Google Scholar 

  41. Judge AD, Robbins M, Tavakoli I, Levi J, Hu L, Fronda A, Ambegia E, Mcclintock K, Maclachlan I (2009) Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice. J Clin Invest 119:661–673

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  42. Zhang Y, Zhang YF, Bryant J, Charles A, Boado RJ, Pardridge WM (2004) Intravenous RNA interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer. Clin Cancer Res 10:3667–3677

    Article  PubMed  CAS  Google Scholar 

  43. Wolfrum C, Shi S, Jayaprakash KN et al (2007) Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat Biotechnol 25:1149–1157

    Article  PubMed  CAS  Google Scholar 

  44. Lundberg M, Wikstrom S, Johansson M (2003) Cell surface adherence and endocytosis of protein transduction domains. Mol Ther 8:143–150

    Article  PubMed  CAS  Google Scholar 

  45. Oishi M, Nagasaki Y, Itaka K et al (2005) Lactosylated poly(ethylene glycol)-siRNA conjugate through acid-labile β-thiopropionate linkage to construct pH-sensitive polyion complex micelles achieving enhanced gene silencing in hepatoma cells. J Am Chem Soc 127:1624–1625

    Article  PubMed  CAS  Google Scholar 

  46. Behr J-P (1997) The proton sponge: a trick to enter cells the viruses did not exploit. CHIMIA 51:34–36

    CAS  Google Scholar 

  47. Stevenson M, Ramos-Perez V, Singh S et al (2008) Delivery of siRNA mediated by histidine-containing reducible polycations. J Control Release 130:46–56

    Article  PubMed  CAS  Google Scholar 

  48. Leng Q, Scaria P, Lu P et al (2008) Systemic delivery of HK Raf-1 siRNA polyplexes inhibits MDA-MB-435 xenografts. Cancer Gene Ther 15:485–495

    Article  PubMed  CAS  Google Scholar 

  49. Kumar VV, Pichon C, Refregiers M et al (2003) Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine mediated membrane fusion at acidic pH. Gene Ther 10:1206–1215

    Article  PubMed  CAS  Google Scholar 

  50. Takae S, Miyata K, Oba M et al (2008) PEG-detachable polyplex micelles based on disulfide-linked block catiomers as bioresponsive nonviral gene vectors. J Am Chem Soc 130:6001–6009

    Article  PubMed  CAS  Google Scholar 

  51. Sahay G, Querbes W, Alabi C, Eltoukhy A, Sarkar S, Zurenko C et al (2013) Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat Biotechnol 31:653–658

    Google Scholar 

  52. Gilleron J, Querbes W, Zeigerer A, Borodovsky A, Marsico G, Schubert U et al (2013) Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat Biotechnol 31:638–646

    Google Scholar 

  53. Pan BT, Johnstone RM (1983) Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 33:967–978

    Article  PubMed  CAS  Google Scholar 

  54. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654–659

    Article  PubMed  CAS  Google Scholar 

  55. Van Den Boorn JG, Schlee M, Coch C, Hartmann G (2011) SiRNA delivery with exosome nanoparticles. Nat Biotechnol 29:325–326

    Article  PubMed  Google Scholar 

  56. Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ (2011) Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol 29:341–345

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Immunology, Institute for Cancer Research, Oslo University Hospital, Montebello, 0310, Oslo, Norway

    Mouldy Sioud

  2. Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Ullernchausseen 70, Montebello, 0310, Oslo, Norway

    Mouldy Sioud

Authors
  1. Mouldy Sioud
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mouldy Sioud .

Editor information

Editors and Affiliations

  1. Institute for Cancer Research, Oslo University Hospital, Montebello, Oslo, Norway

    Mouldy Sioud

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Sioud, M. (2015). RNA Interference: Mechanisms, Technical Challenges, and Therapeutic Opportunities. In: Sioud, M. (eds) RNA Interference. Methods in Molecular Biology, vol 1218. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1538-5_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-1538-5_1

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1537-8

  • Online ISBN: 978-1-4939-1538-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation