Antiviral RNAi pp 293-311 | Cite as

RNAi-Inducing Lentiviral Vectors for Anti-HIV-1 Gene Therapy

  • Ying Poi Liu
  • Jan-Tinus Westerink
  • Olivier ter Brake
  • Ben Berkhout
Part of the Methods in Molecular Biology book series (MIMB, volume 721)


RNA interference (RNAi)-based gene therapy for the treatment of HIV-1 infection provides a novel antiviral approach. For delivery of RNAi inducers to CD4+ T cells or CD34+ blood stem cells, lentiviral vectors are attractive because of their ability to transduce nondividing cells. In addition, lentiviral vectors allow stable transgene expression by inserting their cargo into the host cell genome. However, use of the HIV-1-based lentiviral vector also creates specific problems. The RNAi inducers can target HIV-1 sequences in the genomic RNA of the lentiviral vector. As the RNAi-inducing cassette contains palindromic sequences, the lentiviral vector RNA genome will have a perfect target sequence for the expressed RNAi inducer. Vectors encoding microRNAs face the putative problem that the vector RNA genome can be inactivated by Drosha processing. Here, we describe the design of lentiviral vectors with single or multiple RNAi-inducing antiviral cassettes. The possibility of titer reduction and some effective countermeasures are also presented.

Key words

RNAi Lentiviral vector siRNA shRNA miRNA HIV-1 Gene therapy Antiviral Titer Transduction 


  1. 1.
    Waterhouse, P. M., Wang, M. B., Lough, T. (2001) Gene silencing as an adaptive defence against viruses. Nature 411, 834–42.PubMedCrossRefGoogle Scholar
  2. 2.
    Voinnet, O. (2001) RNA silencing as a plant immune system against viruses. Trends Genet 17, 449–59.PubMedCrossRefGoogle Scholar
  3. 3.
    Wilkins, C., Dishongh, R., Moore, S. C., Whitt, M. A., Chow, M., Machaca, K. (2005) RNA interference is an antiviral defence mechanism in Caenorhabditis elegans. Nature 436, 1044–7.PubMedCrossRefGoogle Scholar
  4. 4.
    Wang, X. H., Aliyari, R., Li, W. X. et al. (2006) RNA interference directs innate immunity against viruses in adult Drosophila. Science 312, 452–4.PubMedCrossRefGoogle Scholar
  5. 5.
    Segers, G. C., Zhang, X., Deng, F., Sun, Q., Nuss, D. L. (2007) Evidence that RNA silencing functions as an antiviral defense mechanism in fungi. Proc Natl Acad Sci U S A 104, 12902–6.PubMedCrossRefGoogle Scholar
  6. 6.
    Bernstein, E., Caudy, A. A., Hammond, S. M., Hannon, G. J. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–6.PubMedCrossRefGoogle Scholar
  7. 7.
    Yi, R., Qin, Y., Macara, I. G., Cullen, B. R. (2003) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17, 3011–6.PubMedCrossRefGoogle Scholar
  8. 8.
    Lund, E., Guttinger, S., Calado, A., Dahlberg, J. E., Kutay, U. (2004) Nuclear export of microRNA precursors. Science 303, 95–8.PubMedCrossRefGoogle Scholar
  9. 9.
    Bartel, D. P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–97.PubMedCrossRefGoogle Scholar
  10. 10.
    Filipowicz, W. (2005) RNAi: the nuts and bolts of the RISC machine. Cell 122, 17–20.PubMedCrossRefGoogle Scholar
  11. 11.
    Haasnoot, J., Westerhout, E. M., Berkhout, B. (2007) RNA interference against viruses: strike and counterstrike. Nat Biotechnol 25, 1435–43.PubMedCrossRefGoogle Scholar
  12. 12.
    Haasnoot, P. C. J., Berkhout, B. (2006) RNA interference: Its use as antiviral therapy. Handbook of Experimental Pharmacology. Heidelberg: Springer, 117–50.Google Scholar
  13. 13.
    Kim, D. H., Rossi, J. J. (2007) Strategies for silencing human disease using RNA interference. Nat Rev Genet 8, 173–84.PubMedCrossRefGoogle Scholar
  14. 14.
    Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., Conklin, D. S. (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16, 948–58.PubMedCrossRefGoogle Scholar
  15. 15.
    Brummelkamp, T. R., Bernards, R., Agami, R. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–3.PubMedCrossRefGoogle Scholar
  16. 16.
    Zeng, Y., Yi, R., Cullen, B. R. (2003) MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc Natl Acad Sci U S A 100, 9779–84.PubMedCrossRefGoogle Scholar
  17. 17.
    Ter Brake, O., Konstantinova, P., Ceylan, M., Berkhout, B. (2006) Silencing of HIV-1 with RNA interference: a multiple shRNA approach. Mol Ther 14, 883–92.PubMedCrossRefGoogle Scholar
  18. 18.
    Ter Brake, O., ‘t Hooft, K., Liu, Y. P., Centlivre, M., von Eije, K. J., Berkhout, B. (2008) Lentiviral vector design for multiple shRNA expression and durable HIV-1 inhibition. Mol Ther 16, 557–64.PubMedCrossRefGoogle Scholar
  19. 19.
    Liu, Y. P., Haasnoot, J., Berkhout, B. (2007) Design of extended short hairpin RNAs for HIV-1 inhibition. Nucleic Acids Res 35, 5683–93.PubMedCrossRefGoogle Scholar
  20. 20.
    Liu, Y. P., Haasnoot, J., Ter Brake, O., Berkhout, B., Konstantinova, P. (2008) Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron. Nucleic Acids Res 36, 2811–24.PubMedCrossRefGoogle Scholar
  21. 21.
    Liu, Y. P., von Eije, K. J., Schopman, N. C. et al. (2009) Combinatorial RNAi against HIV-1 using extended short hairpin RNAs. Mol Ther 17, 1712–23.PubMedCrossRefGoogle Scholar
  22. 22.
    Liu, Y. P., Gruber, J., Haasnoot, J., Konstantinova, P., Berkhout, B. (2009) RNAi-mediated inhibition of HIV-1 by targeting partially complementary viral sequences. Nucleic Acids Res 37, 6194–204.PubMedCrossRefGoogle Scholar
  23. 23.
    Banerjea, A., Li, M. J., Bauer, G. et al. (2003) Inhibition of HIV-1 by lentiviral vector-transduced siRNAs in T lymphocytes differentiated in SCID-hu mice and CD34+ progenitor cell-derived macrophages. Mol Ther 8, 62–71.PubMedCrossRefGoogle Scholar
  24. 24.
    Boden, D., Pusch, O., Lee, F., Tucker, L., Ramratnam, B. (2004) Efficient gene transfer of HIV-1-specific short hairpin RNA into human lymphocytic cells using recombinant adeno-associated virus vectors. Mol Ther 9, 396–402.PubMedCrossRefGoogle Scholar
  25. 25.
    Das, A. T., Brummelkamp, T. R., Westerhout, E. M. et al. (2004) Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J Virol 78, 2601–5.PubMedCrossRefGoogle Scholar
  26. 26.
    Lee, S. K., Dykxhoorn, D. M., Kumar, P. et al. (2005) Lentiviral delivery of short hairpin RNAs protects CD4 T cells from multiple clades and primary isolates of HIV. Blood 106, 818–26.PubMedCrossRefGoogle Scholar
  27. 27.
    Boden, D., Pusch, O., Silbermann, R., Lee, F., Tucker, L., Ramratnam, B. (2004) Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins. Nucleic Acids Res 32, 1154–8.PubMedCrossRefGoogle Scholar
  28. 28.
    Boden, D., Pusch, O., Lee, F., Tucker, L., Ramratnam, B. (2003) Human immunodeficiency virus type 1 escape from RNA interference. J Virol 77, 11531–5.PubMedCrossRefGoogle Scholar
  29. 29.
    Westerhout, E. M., Ooms, M., Vink, M., Das, A. T., Berkhout, B. (2005) HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucleic Acids Res 33, 796–804.PubMedCrossRefGoogle Scholar
  30. 30.
    Harper, S. Q., Staber, P. D., He, X. et al. (2005) RNA interference improves motor and neuropathological abnormalities in a Huntington’s disease mouse model. Proc Natl Acad Sci U S A 102, 5820–5.PubMedCrossRefGoogle Scholar
  31. 31.
    Ralph, G. S., Radcliffe, P. A., Day, D. M. et al. (2005) Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model. Nat Med 11, 429–33.PubMedCrossRefGoogle Scholar
  32. 32.
    Gimeno, R., Weijer, K., Voordouw, A. et al. (2004) Monitoring the effect of gene silencing by RNA interference in human CD34+ cells injected into newborn RAG2-/- ­gammac-/- mice: functional inactivation of p53 in developing T cells. Blood 104, 3886–93.PubMedCrossRefGoogle Scholar
  33. 33.
    Van den Haute, C., Eggermont, K., Nuttin, B., Debyser, Z., Baekelandt, V. (2003) Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in mouse brain. Hum Gene Ther 14, 1799–807.PubMedCrossRefGoogle Scholar
  34. 34.
    Greber, U. F., Fassati, A. (2003) Nuclear import of viral DNA genomes. Traffic 4, 136–43.PubMedCrossRefGoogle Scholar
  35. 35.
    Follenzi, A., Battaglia, M., Lombardo, A., Annoni, A., Roncarolo, M. G., Naldini, L. (2004) Targeting lentiviral vector expression to hepatocytes limits transgene-specific immune response and establishes long-term expression of human antihemophilic factor IX in mice. Blood 103, 3700–9.PubMedCrossRefGoogle Scholar
  36. 36.
    Kordower, J. H., Emborg, M. E., Bloch, J. et al. (2000) Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson’s disease. Science 290, 767–73.PubMedCrossRefGoogle Scholar
  37. 37.
    Miyoshi, H., Smith, K. A., Mosier, D. E., Verma, I. M., Torbett, B. E. (1999) Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors. Science 283, 682–6.PubMedCrossRefGoogle Scholar
  38. 38.
    Naldini, L., Blomer, U., Gallay, P. et al. (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–7.PubMedCrossRefGoogle Scholar
  39. 39.
    Nguyen, T. H., Oberholzer, J., Birraux, J., Majno, P., Morel, P., Trono, D. (2002) Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes. Mol Ther 6, 199–209.PubMedCrossRefGoogle Scholar
  40. 40.
    Kafri, T., Blomer, U., Peterson, D. A., Gage, F. H., Verma, I. M. (1997) Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat Genet 17, 314–7.PubMedCrossRefGoogle Scholar
  41. 41.
    Laufs, S., Guenechea, G., Gonzalez-Murillo, A. et al. (2006) Lentiviral vector integration sites in human NOD/SCID repopulating cells. J Gene Med 8, 1197–207.PubMedCrossRefGoogle Scholar
  42. 42.
    Montini, E., Cesana, D., Schmidt, M. et al. (2006) Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration. Nat Biotechnol 24, 687–96.PubMedCrossRefGoogle Scholar
  43. 43.
    Montini, E., Cesana, D., Schmidt, M. et al. (2009) The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy. J Clin Invest 119, 964–75.PubMedCrossRefGoogle Scholar
  44. 44.
    Cartier, N., Hacein-Bey-Abina, S., Bartholomae, C. C. et al. (2009) Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326, 818–23.PubMedCrossRefGoogle Scholar
  45. 45.
    Anderson, J., Li, M. J., Palmer, B. et al. (2007) Safety and efficacy of a lentiviral vector containing three anti-HIV genes – CCR5 ribozyme, tat-rev siRNA, and TAR decoy – in SCID-hu mouse-derived T cells. Mol Ther 15, 1182–8.PubMedCrossRefGoogle Scholar
  46. 46.
    Henry, S. D., van der, W. P., Metselaar, H. J., Tilanus, H. W., Scholte, B. J., 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.PubMedCrossRefGoogle Scholar
  47. 47.
    Poluri, A., Sutton, R. E. (2007) Titers of HIV-based vectors encoding shRNAs are reduced by a Dicer-dependent mechanism. Mol Ther 16, 378–86.PubMedCrossRefGoogle Scholar
  48. 48.
    Ter Brake, O., Berkhout, B. (2007) Lentiviral vectors that carry anti-HIV shRNAs: problems and solutions. J Gene Med 9, 743–50.PubMedCrossRefGoogle Scholar
  49. 49.
    Zhou, D., Zhang, J., Wang, C. et al. (2009) A method for detecting and preventing negative RNA interference in preparation of lentiviral vectors for siRNA delivery. RNA 15, 732–40.PubMedCrossRefGoogle Scholar
  50. 50.
    Liu, Y. P., Vink, M. A., Westerink, J. T. et al. (2010) Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies. RNA 16, 1328–39.PubMedCrossRefGoogle Scholar
  51. 51.
    Tafer, H., Ameres, S. L., Obernosterer, G. et al. (2008) The impact of target site accessibility on the design of effective siRNAs. Nat Biotechnol 26, 578–83.PubMedCrossRefGoogle Scholar
  52. 52.
    Obernosterer, G., Tafer, H., Martinez, J. (2008) Target site effects in the RNA interference and microRNA pathways. Biochem Soc Trans 36, 1216–9.PubMedCrossRefGoogle Scholar
  53. 53.
    Ameres, S. L., Martinez, J., Schroeder, R. (2007) Molecular basis for target RNA recognition and cleavage by human RISC. Cell 130, 101–12.PubMedCrossRefGoogle Scholar
  54. 54.
    Brown, K. M., Chu, C. Y., Rana, T. M. (2005) Target accessibility dictates the potency of human RISC. Nat Struct Mol Biol 12, 469–70.PubMedCrossRefGoogle Scholar
  55. 55.
    Seppen, J., Rijnberg, M., Cooreman, M. P., Oude Elferink, R. P. (2002) Lentiviral vectors for efficient transduction of isolated primary quiescent hepatocytes. J Hepatol 36, 459–65.PubMedCrossRefGoogle Scholar
  56. 56.
    Kotsopoulou, E., Kim, V. N., Kingsman, A. J., Kingsman, S. M., Mitrophanous, K. A. (2000) A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene. J Virol 74, 4839–52.PubMedCrossRefGoogle Scholar
  57. 57.
    Dull, T., Zufferey, R., Kelly, M. et al. (1998) A third-generation lentivirus vector with a conditional packaging system. J Virol 72, 8463–71.PubMedGoogle Scholar
  58. 58.
    Jeeninga, R. E., Hoogenkamp, M., Armand-Ugon, M., de Baar, M., Verhoef, K., Berkhout, B. (2000) Functional differences between the long terminal repeat transcriptional promoters of HIV-1 subtypes A through G. J Virol 74, 3740–51.PubMedCrossRefGoogle Scholar
  59. 59.
    Yu, J. Y., DeRuiter, S. L., Turner, D. L. (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci U S A 99, 6047–52.PubMedCrossRefGoogle Scholar
  60. 60.
    Koper-Emde, D., Herrmann, L., Sandrock, B., Benecke, B. J. (2004) RNA interference by small hairpin RNAs synthesised under control of the human 7S K RNA promoter. Biol Chem 385, 791–4.PubMedCrossRefGoogle Scholar
  61. 61.
    Denti, M. A., Rosa, A., Sthandier, O., De Angelis, F. G., Bozzoni, I. (2004) A new vector, based on the PolII promoter of the U1 snRNA gene, for the expression of siRNAs in mammalian cells. Mol Ther 10, 191–9.PubMedCrossRefGoogle Scholar
  62. 62.
    Haqqi, T., Zhao, X., Panciu, A., Yadav, S. P. (2002) Sequencing in the presence of betaine: improvement in sequencing of the localized repeat sequence regions. J Biomol Tech 13, 265–71.PubMedGoogle Scholar
  63. 63.
    Andersson, M. G., Haasnoot, P. C. J., Xu, N., Berenjian, S., Berkhout, B., Akusjarvi, G. (2005) Suppression of RNA interference by adenovirus virus-associated RNA. J Virol 79, 9556–65.PubMedCrossRefGoogle Scholar
  64. 64.
    de Vries, W., Haasnoot, J., van der Velden, J. et al. (2008) Increased virus replication in mammalian cells by blocking intracellular innate defense responses. Gene Ther 15, 545–52.PubMedCrossRefGoogle Scholar
  65. 65.
    Haasnoot, J., de Vries, W., Geutjes, E. J., Prins, M., de Haan, P., Berkhout, B. (2007) The Ebola virus VP35 protein is a suppressor of RNA silencing. PLoS Pathog 3, e86.PubMedCrossRefGoogle Scholar
  66. 66.
    Popa, I., Harris, M. E., Donello, J. E., Hope, T. J. (2002) CRM1-dependent function of a cis-acting RNA export element. Mol Cell Biol 22, 2057–67.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2011

Authors and Affiliations

  • Ying Poi Liu
    • 1
  • Jan-Tinus Westerink
  • Olivier ter Brake
  • Ben Berkhout
  1. 1.Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical CenterUniversity of AmsterdamAmsterdamThe Netherlands

Personalised recommendations