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Zinc finger nuclease: a new approach for excising HIV-1 proviral DNA from infected human T cells

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Abstract

A major reason that Acquired Immune Deficiency Syndrome (AIDS) cannot be completely cured is the human immunodeficiency virus 1 (HIV-1) provirus integrated into the human genome. Though existing therapies can inhibit replication of HIV-1, they cannot eradicate it. A molecular therapy gains popularity due to its specifically targeting to HIV-1 infected cells and effectively removing the HIV-1, regardless of viral genes being active or dormant. Now, we propose a new method which can excellently delete the HIV provirus from the infected human T cell genome. First, we designed zinc-finger nucleases (ZFNs) that target a sequence within the long terminal repeat (LTR) U3 region that is highly conserved in whole clade. Then, we screened out one pair of ZFN and named it as ZFN-U3. We discovered that ZFN-U3 can exactly target and eliminate the full-length HIV-1 proviral DNA after the infected human cell lines treated with it, and the frequency of its excision was about 30 % without cytotoxicity. These results prove that ZFN-U3 can effciently excise integrated HIV-1 from the human genome in infected cells. This method to delete full length HIV-1 in human genome can therefore provide a novel approach to cure HIV-infected individuals in the future.

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References

  1. Richman DD, Margolis DM, Delaney M, Greene WC, Hazuda D, Pomerantz RJ (2009) The challenge of finding a cure for HIV infection. Science 323:1304–1307

    Article  CAS  PubMed  Google Scholar 

  2. Katlama C, Deeks SG, Autran B, Martinez-Picado J, van Lunzen J, Rouzioux C, Miller M, Vella S, Schmitz JE, Ahlers J, Richman DD, Sekaly RP (2013) Barriers to a cure for HIV: new ways to target and eradicate HIV-1 reservoirs. Lancet 381:2109–2117

    Article  CAS  PubMed  Google Scholar 

  3. Marcellin F, Spire B, Carrieri MP, Roux P (2013) Assessing adherence to antiretroviral therapy in randomized HIV clinical trials: a review of currently used methods. Expert Rev Anti Infect Ther 11:239–250

    Article  CAS  PubMed  Google Scholar 

  4. Martinez-Colom A, Lasarte S, Fernandez-Pineda A, Relloso M, Munoz-Fernandez MA (2013) A new chimeric protein represses HIV-1 LTR-mediated expression by DNA methylase. Antivir Res 98:394–400

    Article  CAS  PubMed  Google Scholar 

  5. Nisole S, Saïb A (2004) Early steps of retrovirus replicative cycle. Retrovirology 1:9

    Article  PubMed Central  PubMed  Google Scholar 

  6. Chun T-W, Stuyver L, Mizell SB, Ehler LA, Mican JAM, Baseler M, Lloyd AL, Nowak MA, Fauci AS (1997) Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci 94:13193–13197

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Finzi D, Hermankova M, Pierson T, Carruth LM, Buck C, Chaisson RE, Quinn TC, Chadwick K, Margolick J, Brookmeyer R (1997) Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278:1295–1300

    Article  CAS  PubMed  Google Scholar 

  8. Trono D, Van Lint C, Rouzioux C, Verdin E, Barre´-Sinoussi F, Chun T-W, Chomont N (2010) HIV persistence and the prospect of long-term drug-free remissions for HIV-infected individuals. Science 329:174–180

    Article  CAS  PubMed  Google Scholar 

  9. Taube R, Peterlin M (2013) Lost in transcription: molecular mechanisms that control HIV latency. Viruses 5:902–927

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Mussolino C, Morbitzer R, Lutge F, Dannemann N, Lahaye T, Cathomen T (2011) A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res 39:9283–9293

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Schiffer JT, Aubert M, Weber ND, Mintzer E, Stone D, Jerome KR (2012) Targeted DNA mutagenesis for the cure of chronic viral infections. J Virol 86:8920–8936

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Ebina H, Misawa N, Kanemura Y, Koyanagi Y (2013) Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep 3:2510

    Article  PubMed Central  PubMed  Google Scholar 

  13. Sarkar I, Hauber I, Hauber J, Buchholz F (2007) HIV-1 proviral DNA excision using an evolved recombinase. Science 316:1912–1915

    Article  CAS  PubMed  Google Scholar 

  14. Aubert M, Ryu BY, Banks L, Rawlings DJ, Scharenberg AM, Jerome KR (2011) Successful targeting and disruption of an integrated reporter lentivirus using the engineered homing endonuclease Y2 I-AniI. PLoS One 6:e16825

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11:636–646

    Article  CAS  PubMed  Google Scholar 

  16. Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300:764

    Article  CAS  PubMed  Google Scholar 

  17. Lee HJ, Kim E, Kim J-S (2010) Targeted chromosomal deletions in human cells using zinc finger nucleases. Genome Res 20:81–89

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Urnov FD, Miller JC, Lee Y-L, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435:646–651

    Article  CAS  PubMed  Google Scholar 

  19. Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, Wang N, Lee G, Bartsevich VV, Lee Y-L (2008) Establishment of HIV-1 resistance in CD4 + T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 26:808–816

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Holt N, Wang J, Kim K, Friedman G, Wang X, Taupin V, Crooks GM, Kohn DB, Gregory PD, Holmes MC (2010) Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat Biotechnol 28:839–847

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver RC, Katibah GE, Amora R, Boydston EA, Zeitler B (2009) Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 27:851–857

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Doyon Y, McCammon JM, Miller JC, Faraji F, Ngo C, Katibah GE, Amora R, Hocking TD, Zhang L, Rebar EJ (2008) Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol 26:702–708

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Li H, Haurigot V, Doyon Y, Li T, Wong SY, Bhagwat AS, Malani N, Anguela XM, Sharma R, Ivanciu L (2011) In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475:217–221

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Yang D, Yang H, Li W, Zhao B, Ouyang Z, Liu Z, Zhao Y, Fan N, Song J, Tian J (2011) Generation of PPARγ mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning. Cell Res 21:979–982

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Cui X, Ji D, Fisher DA, Wu Y, Briner DM, Weinstein EJ (2010) Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat Biotechnol 29:64–67

    Article  PubMed  Google Scholar 

  26. Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui X, Meng X (2009) Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325:433

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Ochiai H, Sakamoto N, Fujita K, Nishikawa M, Suzuki K-i, Matsuura S, Miyamoto T, Sakuma T, Shibata T, Yamamoto T (2012) Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos. Proc Natl Acad Sci 109:10915–10920

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Merlin C, Beaver LE, Taylor OR, Wolfe SA, Reppert SM (2013) Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases. Genome Res 23:159–168

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Segal DJ, Gonçalves J, Eberhardy S, Swan CH, Torbett BE, Li X, Barbas CF (2004) Attenuation of HIV-1 replication in primary human cells with a designed zinc finger transcription factor. J Biol Chem 279:14509–14519

    Article  CAS  PubMed  Google Scholar 

  30. Eberhardy SR, Goncalves J, Coelho S, Segal DJ, Berkhout B, Barbas CF (2006) Inhibition of human immunodeficiency virus type 1 replication with artificial transcription factors targeting the highly conserved primer-binding site. J Virol 80:2873–2883

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Sakkhachornphop S, Jiranusornkul S, Kodchakorn K, Nangola S, Sirisanthana T, Tayapiwatana C (2009) Designed zinc finger protein interacting with the HIV-1 integrase recognition sequence at 2-LTR-circle junctions. Protein Sci 18:2219–2230

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Sakkhachornphop S, Barbas CF III, Keawvichit R, Wongworapat K, Tayapiwatana C (2012) Zinc finger protein designed to target 2-long terminal repeat junctions interferes with human immunodeficiency virus integration. Hum Gene Ther 23:932–942

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Qu X, Wang P, Ding D, Li L, Wang H, Ma L, Zhou X, Liu S, Lin S, Wang X, Zhang G, Liu L, Wang J, Zhang F, Lu D, Zhu H (2013) Zinc-finger-nucleases mediate specific and efficient excision of HIV-1 proviral DNA from infected and latently infected human T cells. Nucleic Acids Res 41:7771–7782

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Jordan A, Bisgrove D, Verdin E (2003) HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J 22:1868–1877

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Tanaka A, Takeda S, Kariya R, Matsuda K, Urano E, Okada S, Komano J (2013) A novel therapeutic molecule against HTLV-1 infection targeting provirus. Leukemia 27:1621–1627

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by National Natural Science Funding of China (31271418 and 31171247) and the National Grand Program on Key Infectious Disease (2014ZX10001003).

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Authors and Affiliations

  1. State Key Laboratory of Genetic Engineering, School of Life Sciences, Institute of Genetics, Fudan University, Shanghai, 200433, China

    Xiying Qu, Pengfei Wang, Donglin Ding, Xiaohui Wang, Gongmin Zhang, Xin Zhou, Lin Liu, Xiaoli Zhu, Hanxian Zeng & Huanzhang Zhu

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  1. Xiying Qu
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  2. Pengfei Wang
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  3. Donglin Ding
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  4. Xiaohui Wang
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Correspondence to Huanzhang Zhu.

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11033_2014_3456_MOESM1_ESM.tif

Conservation analysis of ZFN-U3 binding sequence and LoxLTR sequence recognized by Tre-recombinase in different HIV-1 subtypes. a The ZFN-U3 binding sequence, located at 408–440 bp within the HXB2 reference isolate (GenBank accession number K03455) (TIFF 5555 kb).

11033_2014_3456_MOESM2_ESM.tif

Conservation analysis of ZFN-U3 binding sequence and LoxLTR sequence recognized by Tre-recombinase in different HIV-1 subtypes. b the LoxLTR sequence, located at 193–226 of the HXB2 reference isolate, were aligned with all HIV-1 genome sequences in the Los Alamos HIV Sequence Database (http://www.hiv.lanl.gov/) using a web alignment tool (http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html). Then the alignments were used to highlight mismatches using the Highlighter for Nucleotide Sequences v2.1.1 online (http://www.hiv.lanl.gov/content/sequence/HIGHLIGHT/HIGHLIGHT_XYPLOT/highlighter.html). Mismatches are represented in different colors: A Green, T Red, G Orange, C Light blue, Gaps-Gray. For ZFN-U3 binding sequences, the total number of analyzed ZFN-U3 binding sequences was 299 and the average similarity was 0.922, while for LoxLTR, the total number of analyzed sequences was 269 and the average similarity was 0.775. (TIFF 4056 kb)

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Qu, X., Wang, P., Ding, D. et al. Zinc finger nuclease: a new approach for excising HIV-1 proviral DNA from infected human T cells. Mol Biol Rep 41, 5819–5827 (2014). https://doi.org/10.1007/s11033-014-3456-3

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  • DOI: https://doi.org/10.1007/s11033-014-3456-3

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