Skip to main content
SpringerLink
Log in

Production of Multicopy shRNA Lentiviral Vectors for Antiviral Therapy

  • Protocol
  • First Online:
Book cover Antiviral RNAi

Part of the book series: Methods in Molecular Biology ((MIMB,volume 721))

Abstract

For effective RNA interference (RNAi)-based therapies against viral infection, particularly highly mutational viruses like HCV and HIV, combinational strategies that target multiple regions within a viral genome are required to prevent resistance. The use of lentiviral vectors for combinatorial RNAi (coRNAi) offers possibilities to deliver multiple short hairpin RNA (shRNA) sequences simultaneously to individual cells while maintaining high expression levels required to suppress viral replication. By applying coRNAi, one can impart either a protective strategy, i.e., treatment prior to infection, or a long-term treatment postinfection without the eventuality of mutational outgrowth due to incomplete selection pressure. In this chapter, we provide a detailed description of the methods available to create coRNAi vectors and discuss some of the current problems and technical limitations.

An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-61779-037-9_26

An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-61779-037-9_26

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. Pan, Q., Henry, S. D., Metselaar, H. J., Scholte, B., Kwekkeboom, J., Tilanus, H. W., Janssen, H. L., and van der Laan, L. J. (2009) Combined antiviral activity of interferon-alpha and RNA interference directed against hepatitis C without affecting vector delivery and gene silencing. J Mol Med 87, 713–22.

    Article  PubMed  CAS  Google Scholar 

  2. Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D., and Naldini, L. (1998) A third-generation lentivirus vector with a conditional packaging system. J Virol 72, 8463–71.

    PubMed  CAS  Google Scholar 

  3. Boden, D., Pusch, O., Lee, F., Tucker, L., and Ramratnam, B. (2003) Human immunodeficiency virus type 1 escape from RNA interference. J Virol 77, 11531–5.

    Article  PubMed  CAS  Google Scholar 

  4. Gitlin, L., Karelsky, S., and Andino, R. (2002) Short interfering RNA confers intracellular antiviral immunity in human cells. Nature 418, 430–4.

    Article  PubMed  CAS  Google Scholar 

  5. Ogata, N., Alter, H. J., Miller, R. H., and Purcell, R. H. (1991) Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. Proc Natl Acad Sci U S A 88, 3392–6.

    Article  PubMed  CAS  Google Scholar 

  6. Okamoto, H., Kojima, M., Okada, S., Yoshizawa, H., Iizuka, H., Tanaka, T., Muchmore, E. E., Peterson, D. A., Ito, Y., and Mishiro, S. (1992) Genetic drift of hepatitis C virus during an 8.2-year infection in a chimpanzee: variability and stability. Virology 190, 894–9.

    Article  PubMed  CAS  Google Scholar 

  7. Young, K. C., Lindsay, K. L., Lee, K. J., Liu, W. C., He, J. W., Milstein, S. L., and Lai, M. M. (2003) Identification of a ribavirin-resistant NS5B mutation of hepatitis C virus during ribavirin monotherapy. Hepatology 38, 869–78.

    PubMed  CAS  Google Scholar 

  8. Kanda, T., Yokosuka, O., Imazeki, F., Tanaka, M., Shino, Y., Shimada, H., Tomonaga, T., Nomura, F., Nagao, K., Ochiai, T., and Saisho, H. (2004) Inhibition of subgenomic hepatitis C virus RNA in Huh-7 cells: ribavirin induces mutagenesis in HCV RNA. J Viral Hepat 11, 479–87.

    Article  PubMed  CAS  Google Scholar 

  9. Konishi, M., Wu, C. H., Kaito, M., Hayashi, K., Watanabe, S., Adachi, Y., and Wu, G. Y. (2006) siRNA-resistance in treated HCV replicon cells is correlated with the development of specific HCV mutations. J Viral Hepat 13, 756–61.

    Article  PubMed  Google Scholar 

  10. Wilson, J. A., and Richardson, C. D. (2005) Hepatitis C virus replicons escape RNA interference induced by a short interfering RNA directed against the NS5b coding region. J Virol 79, 7050–8.

    Article  PubMed  CAS  Google Scholar 

  11. Henry, S. D., van der Wegen, P., Metselaar, H. J., Tilanus, H. W., Scholte, B. J., and van der Laan, L. J. (2006) Simultaneous targeting of HCV replication and viral binding with a single lentiviral vector containing multiple RNA interference expression cassettes. Mol Ther 14, 485–93.

    Article  PubMed  CAS  Google Scholar 

  12. Korf, M., Jarczak, D., Beger, C., Manns, M. P., and Kruger, M. (2005) Inhibition of hepatitis C virus translation and subgenomic replication by siRNAs directed against highly conserved HCV sequence and cellular HCV cofactors. J Hepatol 43, 225–34.

    Article  PubMed  CAS  Google Scholar 

  13. Novina, C. D., Murray, M. F., Dykxhoorn, D. M., Beresford, P. J., Riess, J., Lee, S. K., Collman, R. G., Lieberman, J., Shankar, P., and Sharp, P. A. (2002) siRNA-directed inhibition of HIV-1 infection. Nat Med 8, 681–6.

    PubMed  CAS  Google Scholar 

  14. Ciesek, S., Steinmann, E., Wedemeyer, H., Manns, M. P., Neyts, J., Tautz, N., Madan, V., Bartenschlager, R., von Hahn, T., and Pietschmann, T. (2009) Cyclosporine A inhibits hepatitis C virus nonstructural protein 2 through cyclophilin A. Hepatology 50, 1638–45.

    Article  PubMed  CAS  Google Scholar 

  15. McIntyre, G. J., Groneman, J. L., Tran, A., and Applegate, T. L. (2008) An infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA. PLoS ONE 3, e3827.

    Article  PubMed  Google Scholar 

  16. McIntyre, G. J., Yu, Y. H., Tran, A., Jaramillo, A. B., Arndt, A. J., Millington, M. L., Boyd, M. P., Elliott, F. A., Shen, S. W., Murray, J. M., and Applegate, T. L. (2009) Cassette deletion in multiple shRNA lentiviral vectors for HIV-1 and its impact on treatment success. Virol J 6, 184.

    Article  PubMed  Google Scholar 

  17. Brake, O. T., Hooft, K., Liu, Y. P., Centlivre, M., Jasmijn von Eije, K., and Berkhout, B. (2008) Lentiviral vector design for multiple shRNA expression and durable HIV-1 inhibition. Mol Ther 16, 557–64.

    Article  PubMed  Google Scholar 

  18. Gou, D., Weng, T., Wang, Y., Wang, Z., Zhang, H., Gao, L., Chen, Z., Wang, P., and Liu, L. (2007) A novel approach for the construction of multiple shRNA expression vectors. J Gene Med 9, 751–63.

    Article  PubMed  CAS  Google Scholar 

  19. Weinberg, M. S., Ely, A., Barichievy, S., Crowther, C., Mufamadi, S., Carmona, S., and Arbuthnot, P. (2007) Specific inhibition of HBV replication in vitro and in vivo with expressed long hairpin RNA. Mol Ther 15, 534–41.

    Article  PubMed  CAS  Google Scholar 

  20. Sano, M., Li, H., Nakanishi, M., and Rossi, J. J. (2008) Expression of long anti-HIV-1 hairpin RNAs for the generation of multiple siRNAs: advantages and limitations. Mol Ther 16, 170–7.

    Article  PubMed  CAS  Google Scholar 

  21. Ter Brake, O., and Berkhout, B. (2007) Lentiviral vectors that carry anti-HIV shRNAs: problems and solutions. J Gene Med 9, 743–50.

    Article  PubMed  Google Scholar 

  22. Konstantinova, P., de Vries, W., Haasnoot, J., Ter Brake, O., de Haan, P., and Berkhout, B. (2006) Inhibition of human immunodeficiency virus type 1 by RNA interference using long-hairpin RNA. Gene Ther 13, 1403–13.

    Article  PubMed  CAS  Google Scholar 

  23. Saayman, S., Barichievy, S., Capovilla, A., Morris, K. V., Arbuthnot, P., and Weinberg, M. S. (2008) The efficacy of generating three independent anti-HIV-1 siRNAs from a single U6 RNA Pol III-expressed long hairpin RNA. PLoS ONE 3, e2602.

    Article  PubMed  Google Scholar 

  24. Liu, Y. P., von Eije, K. J., Schopman, N. C., Westerink, J. T., ter Brake, O., Haasnoot, J., and Berkhout, B. (2009) Combinatorial RNAi against HIV-1 using extended short hairpin RNAs. Mol Ther 17, 1712–23.

    Article  PubMed  CAS  Google Scholar 

  25. Barichievy, S., Saayman, S., von Eije, K. J., Morris, K. V., Arbuthnot, P., and Weinberg, M. S. (2007) The inhibitory efficacy of RNA POL III-expressed long hairpin RNAs targeted to untranslated regions of the HIV-1 5′ long terminal repeat. Oligonucleotides 17, 419–31.

    Article  PubMed  CAS  Google Scholar 

  26. Pan, Q., Tilanus, H. W., Janssen, H. L., and van der Laan, L. J. W. (2009) Prospects of RNAi and microRNA-based therapies for hepatitis C. Expert Opin Biol Ther 9, 713–24.

    Article  PubMed  CAS  Google Scholar 

  27. Pan, Q., Henry, S. D., Scholte, B. J., Tilanus, H. W., Janssen, H. L., and van der Laan, L. J. W. (2007) New therapeutic opportunities for Hepatitis C based on small RNA. World J Gastroenterol 13, 4431–6.

    PubMed  CAS  Google Scholar 

  28. Aagaard, L. A., Zhang, J., von Eije, K. J., Li, H., Saetrom, P., Amarzguioui, M., and Rossi, J. J. (2008) Engineering and optimization of the miR-106b cluster for ectopic expression of multiplexed anti-HIV RNAs. Gene Ther 15, 1536–49.

    Article  PubMed  CAS  Google Scholar 

  29. Boden, D., Pusch, O., Silbermann, R., Lee, F., Tucker, L., and Ramratnam, B. (2004) Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins. Nucleic Acids Res 32, 1154–8.

    Article  PubMed  CAS  Google Scholar 

  30. Cullen, B. R. (2006) Viruses and microRNAs. Nat Genet 38 Suppl 1, S25–30.

    Article  PubMed  CAS  Google Scholar 

  31. Shan, Z. X., Lin, Q. X., Yang, M., Deng, C. Y., Kuang, S. J., Zhou, Z. L., Xiao, D. Z., Liu, X. Y., Lin, S. G., and Yu, X. Y. (2009) A quick and efficient approach for gene silencing by using triple putative microRNA-based short hairpin RNAs. Mol Cell Biochem 323, 81–9.

    Article  PubMed  CAS  Google Scholar 

  32. McManus, M. T., Petersen, C. P., Haines, B. B., Chen, J., and Sharp, P. A. (2002) Gene silencing using micro-RNA designed hairpins. RNA 8, 842–50.

    Article  PubMed  CAS  Google Scholar 

  33. Shan, Z., Lin, Q., Deng, C., Li, X., Huang, W., Tan, H., Fu, Y., Yang, M., and Yu, X. Y. (2009) An efficient method to enhance gene silencing by using precursor microRNA designed small hairpin RNAs. Mol Biol Rep 36, 1483–9.

    Article  PubMed  CAS  Google Scholar 

  34. Shin, K. J., Wall, E. A., Zavzavadjian, J. R., Santat, L. A., Liu, J., Hwang, J. I., Rebres, R., Roach, T., Seaman, W., Simon, M. I., and Fraser, I. D. (2006) A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression. Proc Natl Acad Sci U S A 103, 13759–64.

    Article  PubMed  CAS  Google Scholar 

  35. Sun, D., Melegari, M., Sridhar, S., Rogler, C. E., and Zhu, L. (2006) Multi-miRNA hairpin method that improves gene knockdown efficiency and provides linked multi-gene knockdown. Biotechniques 41, 59–63.

    Article  PubMed  CAS  Google Scholar 

  36. Zhu, X., Santat, L. A., Chang, M. S., Liu, J., Zavzavadjian, J. R., Wall, E. A., Kivork, C., Simon, M. I., and Fraser, I. D. (2007) A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs. BMC Mol Biol 8, 98.

    Article  PubMed  Google Scholar 

  37. Lim, L. P., Lau, N. C., Garrett-Engele, P., Grimson, A., Schelter, J. M., Castle, J., Bartel, D. P., Linsley, P. S., and Johnson, J. M. (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–73.

    Article  PubMed  CAS  Google Scholar 

  38. Dinh, A., and Mo, Y. Y. (2005) Alternative approach to generate shRNA from cDNA. Biotechniques 38, 629–32.

    Article  PubMed  CAS  Google Scholar 

  39. Wu, H., Dinh, A., and Mo, Y. Y. (2007) Generation of shRNAs from randomized oligonucleotides. Biol Proced Online 9, 9–17.

    Article  PubMed  CAS  Google Scholar 

  40. Lizee, G., Aerts, J. L., Gonzales, M. I., Chinnasamy, N., Morgan, R. A., and Topalian, S. L. (2003) Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression. Hum Gene Ther 14, 497–507.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Dr. Bob Scholte, Prof. Hugo Tilanus Prof. Herold Metselaar, and Prof. Harry Janssen for general support. This study has been supported financially by the Erasmus MC Translational Research Fund and the Liver Research Foundation (SLO) Rotterdam.

Author information

Authors and Affiliations

  1. Department of Surgery, Erasmus MC – University Medical Center, Rotterdam, The Netherlands

    Luc J. W. van der Laan

Authors
  1. Scot D. Henry
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Quiwei Pan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Luc J. W. van der Laan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Luc J. W. van der Laan .

Editor information

Editors and Affiliations

  1. Inst. for Infection, Inflammation & Immu, Dept. Medical Microbiology, Radboud Univ.Nijmegen Medical Ctr, Geert Grooteplein 28, Nijmegen, 6525 GA, Netherlands

    Ronald P. van Rij

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Humana Press

About this protocol

Cite this protocol

Henry, S.D., Pan, Q., van der Laan, L.J.W. (2011). Production of Multicopy shRNA Lentiviral Vectors for Antiviral Therapy. In: van Rij, R. (eds) Antiviral RNAi. Methods in Molecular Biology, vol 721. Humana Press. https://doi.org/10.1007/978-1-61779-037-9_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-037-9_19

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-036-2

  • Online ISBN: 978-1-61779-037-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation