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Complete knockdown of CCR5 by lentiviral vector-expressed siRNAs and protection of transgenic macrophages against HIV-1 infection

Abstract

The CCR5 co-receptor is necessary for cellular entry by R5 tropic viral strains involved in primary HIV infection, but is dispensable for normal human physiology. Owing to its crucial role in HIV-1 infection, the CCR5 co-receptor has been the subject of many therapeutic approaches, including gene therapy. siRNA targeting was shown to be effective in downregulating CCR5 expression and conferring significant protection against HIV-1 in susceptible cells. However, complete knockdown of CCR5 expression has not been achieved and thus remains an elusive goal. In these studies, we identified new CCR5 siRNAs capable of achieving complete knockdown of the co-receptor expression. Our transfection studies have shown that longer 28-mer short hairpin siRNAs are very effective in gene downregulation as assessed by fluorescence-activated cell sorting and transcript quantitation by quantitative real-time polymerase chain reaction. These siRNAs conferred strong antiviral protection during viral challenge. To obtain stable expression, highly potent siRNA expression cassettes were introduced into lentiviral vectors. Similar high levels of CCR5 downregulation were observed in stably transduced cells with concomitant viral protection in cultured cell lines. To translate these results to a stem cell gene therapy setting, CD34 hematopoietic progenitor cells were transduced with lentiviral vectors to derive transgenic macrophages. The transgenic cells also exhibited high levels of CCR5 downregulation and viral resistance. With regard to Pol-III promoter-mediated siRNA expression, higher efficacies were obtained with U6-driven CCR5 siRNAs. However, in contrast to previous reports, no apparent cytotoxicities were observed in transgenic cells containing U6-driven siRNA constructs. Thus the above anti-CCR5 siRNAs are among the most effective demonstrated to date and are very promising candidates for clinical applications.

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References

  1. Martinez-Picado J, DePasquale MP, Kartsonis N, Hanna GJ, Wong J, Finzi D et al. Antiretroviral resistance during successful therapy of HIV type 1 infection. Proc Natl Acad Sci USA 2000; 97: 10948–10953.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Lafeuillade A, Poggi C, Hittinger G, Chadapaud S . Phenotypic and genotypic resistance to nucleoside reverse transcriptase inhibitors in HIV-1 clinical isolates. HIV Med 2001; 2: 231–235.

    Article  CAS  PubMed  Google Scholar 

  3. Akkina R, Banerjea A, Bai J, Anderson J, Li MJ, Rossi J . siRNAs, ribozymes, and RNA decoys in modeling stem cell-based gene therapy for HIV/AIDS. Anticancer Res 2003; 23: 1997–2006.

    CAS  PubMed  Google Scholar 

  4. Cagnon L, Rossi J . Down regulation of the CCR5 beta-chemokine receptor and inhibition of HIV-1 infection by stable VA1-ribozyme chimeric transcripts. Anti Nucl Acid Drug Dev 2000; 10: 251–261.

    Article  CAS  Google Scholar 

  5. Bai J, Gorantla S, Banda N, Cagnon L, Rossi J, Akkina R . Characterization of anti- CCR5 ribozyme-transduced CD34+ hematopoietic progenitor cells in vitro and in a SCID-hu mouse model in vivo. Mol Ther 2000; 1: 244–254.

    Article  CAS  PubMed  Google Scholar 

  6. Bai J, Rossi J, Akkina R . Multivalent anti-CCR5 ribozymes for stem cell-based HIV type 1 gene therapy. AIDS Res Hum Retroviruses 2001; 17: 385–399.

    Article  CAS  PubMed  Google Scholar 

  7. Bai J, Banda N, Lee NS, Rossi J, Akkina R . RNA-based anti-HIV-1 gene therapeutic constructs in SCID-hu mouse model. Mol Ther 2002; 6: 770–782.

    Article  CAS  PubMed  Google Scholar 

  8. Ding SF, Lombardi R, Nazari R, Joshi S . A combination anti-HIV-1 gene therapy approach using a single transcription unit that expresses antisense, decoy, and sense RNAs, and transdominant negative mutant Gag and Env proteins. Front Biosci 2002; 7: a15–a28.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  11. Sharp P . RNA interference-2001. Genes Dev 2001; 15: 485–490.

    Article  CAS  PubMed  Google Scholar 

  12. Lee NS, Dohjima T, Bauer G, Li H, Li MJ, Ehsani A et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 2002; 20: 500–505.

    Article  CAS  PubMed  Google Scholar 

  13. Novina CD, Murray MF, Dykxhoorn DM, Beresford PJ, Riess J, Lee SK et al. siRNA-directed inhibition of HIV-1 infection. Nat Med 2002; 8: 681–686.

    Article  CAS  PubMed  Google Scholar 

  14. Jacque J, Triques K, Stevenson M . Modulation of HIV-1 replication by RNA interference. Nature 2002; 418: 435–438.

    Article  CAS  PubMed  Google Scholar 

  15. Coburn GA, Cullen BR . Potent and specific inhibition of human immunodeficiency virus type-1 replication by RNA interference. J Virol 2002; 76: 9225–9231.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Capodici J, Kariko K, Weissman D . Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference. J Immunol 2002; 169: 5196–5201.

    Article  PubMed  Google Scholar 

  17. Banerjea A, Li MJ, Bauer G, Remling L, Lee NS, Rossi J et al. Inhibition of HIV-1 by lentiviral vector-transduced siRNAs in lymphocytes differentiated in SCID-hu mice and CD34+ progenitor cell-derived macrophages. Mol Ther 2003; 8: 62–71.

    Article  CAS  PubMed  Google Scholar 

  18. Li M, Bauer G, Michienzi A, Yee JK, Lee NS, Kim J et al. Inhibition of HIV-1 infection by lentiviral vectors expressing Pol III-promoted anti-HIV RNAs. Mol Ther 2003; 8: 196–206.

    Article  CAS  PubMed  Google Scholar 

  19. Haasnoot PCJ, Cupac D, Berkhout B . Inhibition of virus replication by RNA interference. J Biomed Sci 2003; 10: 607–616.

    Article  PubMed  Google Scholar 

  20. Lee MM, Coburn G, McClure MO, Cullen BR . Inhibition of human immunodeficiency virus type 1 replication in primary macrophages by using tat- or CCR5-specific small interfering RNAs expressed from a lentivirus vector. J Virol 2003; 77: 11964–11972.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Anderson J, Banerjea A, Planelles V, Akkina R . Potent suppression of HIV type 1 infection by a short hairpin anti-CXCR4 siRNA. AIDS Res Hum Retroviruses 2003; 19: 699–706.

    Article  CAS  PubMed  Google Scholar 

  22. Anderson J, Banerjea A, Akkina R . Bispecific short hairpin siRNA constructs targeted to CD4, CXCR4, and CCR5 confer HIV-1 resistance. Oligonucleotides 2003; 13: 303–312.

    Article  CAS  PubMed  Google Scholar 

  23. Martinez MA, Gutierrez A, Armand-Ugon M, Blanco J, Parera M, Gomez J et al. Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication. AIDS 2002; 16: 2385–2390.

    Article  CAS  PubMed  Google Scholar 

  24. Qin X, An DS, Chen ISY, Baltimore D . Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci USA 2003; 100: 183–188.

    Article  CAS  PubMed  Google Scholar 

  25. Song E, Lee SK, Dykxhoorn DM, Novina C, Zhang D, Crawford K et al. Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages. J Virol 2003; 77: 7174–7181.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Butticaz C, Ciuffi A, Munoz M, Thomas J, Bridge A, Pebernard S et al. Protection from HIV-1 infection of primary CD4 T cells by CCR5 silencing is effective for the full spectrum of CCR5 expression. Antiviral Ther 2003; 8: 373–377.

    CAS  Google Scholar 

  27. Cordelier P, Morse B, Strayer DS . Targeting CCR5 with siRNAs: using recombinant SV40-derived vectors to protect macrophages and Microglia from R5-Tropic HIV. Oligonucleotides 2003; 13: 281–294.

    Article  CAS  PubMed  Google Scholar 

  28. Zhou N, Fang J, Mukhtar M, Acheampong E, Pomerantz RJ . Inhibition of HIV-1 fusion with small interfering RNAs targeting the chemokine coreceptor CXCR4. Gene Ther 2004; 11: 1703–1712.

    Article  CAS  PubMed  Google Scholar 

  29. Anderson J, Akkina R . HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector. AIDS Res Ther 2005; 2: 1–12.

    Article  PubMed Central  PubMed  Google Scholar 

  30. Anderson J, Akkina R . CXCR4 and CCR5 shRNA transgenic CD34+ cell derived macrophages are functionally normal and resist HIV-1 infection. Retrovirology 2005; 2: 53.

    Article  PubMed Central  PubMed  Google Scholar 

  31. Li MJ, Kim J, Li S, Zaia J, Yee JK, Anderson J et al. Long-term inhibition of HIV-1 infection in primary hematopoietic cells by lentiviral vector delivery of a triple combination of anti-HIV shRNA, anti-CCR5 ribozyme, and a nucleolar-localizing TAR decoy. Mol Ther 2005; 12: 900–909.

    Article  CAS  PubMed  Google Scholar 

  32. Liang WS, Maddukuri A, Teslovich TM, de la Fuente C, Agbottah E, Dadgar S et al. Therapeutic targets for HIV-1 infection in the host proteome. Retrovirology 2005; 2: 20.

    Article  PubMed Central  PubMed  Google Scholar 

  33. Boden D, Pusch O, Lee F, Tucker L, Ramratnam B . Human immunodeficiency virus type 1 escape from RNA interference. J Virol 2003; 77: 11531–11535.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Das AT, Brummelkamp TR, Westerhout EM, Vink M, Madiredjo M, Bernards R et al. Human Immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J Virol 2004; 78: 2601–2605.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Bieniasz PD, Cullen BR . Chemokine receptors and Human Immunodeficiency Virus infection. Front Biosci 1998; 3: 44–58.

    Article  Google Scholar 

  36. Berger EA, Murphy PM, Farber JM . Chemokine receptors as HIV coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol 1999; 17: 657–700.

    Article  CAS  PubMed  Google Scholar 

  37. Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R et al. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply exposed individuals to HIV-1 infection. Cell 1996; 86: 267–377.

    Google Scholar 

  38. Huang Y, Paxton WA, Wolinsky SM, Neumann AU, Zhang L, He T et al. The role of a mutant CCR5 allele in HIV-1 transmission and disease progression. Nat Med 1996; 2: 1240–1243.

    Article  CAS  PubMed  Google Scholar 

  39. Naif HM, Cunningham AL, Alali M, Li S, Nasr N, Buhler MM et al. A human immunodeficiency virus type 1 isolate from an infected person homozygous for CCR5Δ32 exhibits dual tropism by infecting macrophages and MT2 cells via CXCR4. J Virol 2002; 76: 3114–3124.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A . Rational siRNA design for RNA interference. Nat Biotechnol 2004; 3: 326–330.

    Article  Google Scholar 

  41. Boese Q, Leake D, Reynolds A, Read S, Scaringe S, Marshall WS et al. Mechanistic insights aid computational short interfering RNA design. Methods Enzymol 2005; 392: 73–96.

    Article  CAS  PubMed  Google Scholar 

  42. Vermeulen A, Behlen L, Reynolds A, Wolfson A, Marshall WS . The contributions of dsRNA structure to Dicer specificity and efficiency. RNA 2005; 5: 674–682.

    Article  Google Scholar 

  43. Naldini L . In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996; 272: 263–267.

    Article  CAS  PubMed  Google Scholar 

  44. An DS, Koyanagi Y, Zhao JQ, Akkina R, Bristol G, Yamamoto N et al. High-efficiency transduction of human lymphoid progenitor cells and expression in differentiated T cells. J Virol 1997; 71: 1397–1404.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Ailles LE, Naldini L . HIV-1 Derived Lentiviral Vectors. In: Trono D (ed). Lentiviral Vectors. Springer: Berlin, 2002, pp 31–48.

    Chapter  Google Scholar 

  46. Yam P, Li S, Wu J, Hu J, Zaia J, Yee J . Design of HIV-1 vectors for efficient gene delivery into human hematopoietic cells. Mol Ther 2002; 6: 770–782.

    Article  Google Scholar 

  47. Mautino MR, Morgan RA . Gene therapy of HIV-1 infection using lentiviral vectors expressing anti-HIV-1 genes. AIDS Patient Care STDs 2002; 16: 11–26.

    Article  PubMed  Google Scholar 

  48. Siolas D, Lerner C, Burchard J, Ge W, Linsley PS, Paddison PJ et al. Synthetic shRNAs as potent RNAi triggers. Nat Biotech 2004; 23: 227–231.

    Article  Google Scholar 

  49. Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC et al. Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc Natl Acad Sci USA 2004; 101: 1892–1897.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  50. Fedorov Y, Anderson EM, Birmingham A, Reynolds A, Karpilow J, Robinson K et al. Off-target effects by siRNA can induce toxic phenotype. RNA 2006; 7: 1188–1196.

    Article  Google Scholar 

  51. An DS, Qin X-F, Auyeung VC, Mao SH, Kung SKP, Baltimore D et al. Optimization and functional effects of stable short hairpin RNA expression in primary human lymphocytes via lentiviral vectors. Mol Ther 2006; 14: 494–504.

    Article  CAS  PubMed  Google Scholar 

  52. Castanotto D, Li H, Rossi J . Functional siRNA expression from transfected PCR products. RNA 2002; 8: 1454–1460.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgements

The work was supported by NIH grants AI50492 and AI057066 to RA. It has also been facilitated by the infrastructure and resources provided by the Colorado Center for AIDS Research Grant P30 AI054907. We gratefully acknowledge the help of Jon Karpilow and Annaleen Vermeulen of Dharmacon with the design of CCR5 siRNAs and for critically reading the manuscript. We thank Karen Helms for help with FACS sorting and Leila Remling for purification of CD34 cells. NIH AIDS Research and Reference Reagents Program provided many reagents and cell lines used in this work.

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Correspondence to R Akkina.

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Anderson, J., Akkina, R. Complete knockdown of CCR5 by lentiviral vector-expressed siRNAs and protection of transgenic macrophages against HIV-1 infection. Gene Ther 14, 1287–1297 (2007). https://doi.org/10.1038/sj.gt.3302958

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