RNA Interference pp 123-136

Part of the Methods in Molecular Biology book series (MIMB, volume 623)

Strategies in Designing Multigene Expression Units to Downregulate HIV-1



The treatment of viral diseases such as HIV and HCV is limited by the genetic diversity of the viruses, especially when they are under the selective pressure of drugs. This problem holds true for gene-based therapies using RNAi in which there is evolution of drug-resistant strains under the discriminating pressure of treatment (1, 2). In a gene therapy setting for treatment of HIV, the incorporation of multiple effector molecules, targeting different viral and cellular sequences, can improve viral inhibition by substantially delaying the emergence of escape mutants (3-8). However, for short hairpin RNA triggers of RNAi, high levels of expression by strong Pol III promoters has led to cell toxicity, and even death in experimental animals (9, 10). Here, we describe a new combinatorial anti-HIV gene expression system allowing simultaneous expression of multiple RNAi effector units from a single Pol II polycistronic transcript. Our platform is suitable for the inclusion of any shRNA sequence and can be combined with other types of small RNA antiviral inhibitors.

Key words

RNAi HIV-1 miRNAs siRNAs Polycistron 


  1. 1.
    Dykxhoorn, D.M. and Lieberman, J. (2006) Silencing viral infection. PLoS Med 3(7):e242.CrossRefPubMedGoogle Scholar
  2. 2.
    Grimm, D. and Kay, M.A. (2007) Combinatorial RNAi: A Winning Strategy for the Race Against Evolving Targets? Mol. Ther. 15(5): 878–888.PubMedGoogle Scholar
  3. 3.
    Andersson, M.G., Haasnoot, P.C., Xu, N., Berenjian, S., Berkhout, B. and Akusjarvi, G. (2005) Suppression of RNA interference by adenovirus virus-associated RNA. J. Virol. 79(15):9556–9565.CrossRefPubMedGoogle Scholar
  4. 4.
    Chang, L.J., Liu, X. and He, J. (2005) Lentiviral siRNAs targeting multiple highly conserved RNA sequences of human immunodeficiency virus type 1. Gene Ther. 12(14):1133–1144.CrossRefPubMedGoogle Scholar
  5. 5.
    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(4):485–493.CrossRefPubMedGoogle Scholar
  6. 6.
    Li, M. and Rossi, J.J. (2005) Lentiviral vector delivery of siRNA and shRNA encoding genes into cultured and primary hematopoietic cells. Methods Mol. Biol. 309:261–272.PubMedGoogle Scholar
  7. 7.
    ter Brake, O., Konstantinova, P., Ceylan, M. and Berkhout, B. (2006) Silencing of HIV-1 with RNA interference: a multiple shRNA approach. Mol. Ther. 14(6):883–892.CrossRefPubMedGoogle Scholar
  8. 8.
    ter Brake, O., t Hooft, K., Liu, Y.P., Centlivre, M., von Eije, K.J. and Berkhout, B. (2008) Lentiviral vector design for multiple shRNA expression and durable HIV-1 inhibition. Mol. Ther. 16(3):557–564.CrossRefPubMedGoogle Scholar
  9. 9.
    An, D.S., Qin, F.X., Auyeung, V.C., et al. (2006) Optimization and functional effects of stable short hairpin RNA expression in primary human lymphocytes via lentiviral vectors. Mol. Ther. 14(4):494–504.CrossRefPubMedGoogle Scholar
  10. 10.
    Grimm, D., Streetz, K.L., Jopling, C.L., et al. (2006) Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441(7092):537–541.CrossRefPubMedGoogle Scholar
  11. 11.
    Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411(6836):494–498.CrossRefPubMedGoogle Scholar
  12. 12.
    Brummelkamp, T.R., Bernards, R., and Agami, R. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296(5567):550–553.CrossRefPubMedGoogle Scholar
  13. 13.
    Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J., and Conklin, D.S. (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16(8):948–958.CrossRefPubMedGoogle Scholar
  14. 14.
    Bartel, D.P. and Chen, C.Z. (2004) Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs. Nat. Rev. Genet. 5(5):396–400.CrossRefPubMedGoogle Scholar
  15. 15.
    Zeng, Y., Wagner, E.J. and Cullen, B.R. (2002) Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9(6):1327–1333.CrossRefPubMedGoogle Scholar
  16. 16.
    Li, M.J., Bauer, G., Michienzi, A., et al. (2003) Inhibition of HIV-1 infection by lentiviral vectors expressing Pol III-promoted anti-HIV RNAs. Mol. Ther. 8(2):196–206.CrossRefPubMedGoogle Scholar
  17. 17.
    Rubinson, D.A., Dillon, C.P., Kwiatkowski, A.V., et al. (2003) A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33(3): 401–406.CrossRefPubMedGoogle Scholar
  18. 18.
    Snove, O., Jr. and Rossi, J.J. (2006) Expressing short hairpin RNAs in vivo. Nat. Methods 3(9):689–695.CrossRefPubMedGoogle Scholar
  19. 19.
    Song, E., Lee, S.K., Dykxhoorn, D.M., et al. (2003) Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages. J. Virol. 77(13):7174–1781.CrossRefPubMedGoogle Scholar
  20. 20.
    Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297.CrossRefPubMedGoogle Scholar
  21. 21.
    Kim, V.N. (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat. Rev. Mol. Cell Biol. 6(5):376–385.CrossRefPubMedGoogle Scholar
  22. 22.
    Kim, V.N. (2005) Small RNAs: classification, biogenesis, and function. Mol. Cells 19 (1):1–15.CrossRefPubMedGoogle Scholar
  23. 23.
    Aagaard, L.A., Zhang, J., von Eije, K.J., et al. (2008) Engineering and optimization of the miR-106b cluster for ectopic expression of multiplexed anti-HIV RNAs. Gene Ther. 15(23):1536–1549.CrossRefPubMedGoogle Scholar
  24. 24.
    Chendrimada, T.P., Gregory, R.I., Kumaraswamy, E., et al. (2005) TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436(7051):740-744CrossRefPubMedGoogle Scholar
  25. 25.
    Gregory, R.I., Chendrimada, T.P., Cooch, N. and Shiekhattar, R. (2005) Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123(4):631–640.CrossRefPubMedGoogle Scholar
  26. 26.
    Lee, Y., Jeon, K., Lee, J.T., Kim, S. and Kim, V.N. (2002) MicroRNA maturation: stepwise processing and subcellular localization. EMBO J. 21(17):4663–4670.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Beckman Research Institute of City of HopeDuarteUSA

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