Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Dykxhoorn, D.M. and Lieberman, J. (2006) Silencing viral infection. PLoS Med 3(7):e242.
Grimm, D. and Kay, M.A. (2007) Combinatorial RNAi: A Winning Strategy for the Race Against Evolving Targets? Mol. Ther. 15(5): 878–888.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Snove, O., Jr. and Rossi, J.J. (2006) Expressing short hairpin RNAs in vivo. Nat. Methods 3(9):689–695.
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.
Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297.
Kim, V.N. (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat. Rev. Mol. Cell Biol. 6(5):376–385.
Kim, V.N. (2005) Small RNAs: classification, biogenesis, and function. Mol. Cells 19 (1):1–15.
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.
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):740-744
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.
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Zhang, J., Rossi, J.J. (2010). Strategies in Designing Multigene Expression Units to Downregulate HIV-1. In: Min, WP., Ichim, T. (eds) RNA Interference. Methods in Molecular Biology, vol 623. Humana Press. https://doi.org/10.1007/978-1-60761-588-0_8
Download citation
DOI: https://doi.org/10.1007/978-1-60761-588-0_8
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-587-3
Online ISBN: 978-1-60761-588-0
eBook Packages: Springer Protocols