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‘Mini’ U6 Pol III promoter exhibits nucleosome redundancy and supports multiplexed coupling of CRISPR/Cas9 effects

Gene Therapy volume 27, pages 451–458 (2020)Cite this article

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Abstract

RNA polymerase III (Pol III) promoters express short non-coding RNAs and have been adopted for expression of microRNA, interference RNA, and CRISPR single guide RNA (sgRNA). Vectors incorporating H1 and U6 Pol III promoters are being applied for therapeutic genome editing, including multiplexed CRISPR/Cas9 effects. We report a nucleosome-depleted, minimal U6 promoter, which when embedded within lentiviral long terminal repeat (LTR) regions, supports high level transcriptional activity. Furthermore, duplex minimal H1 & U6 promoters transcribed dual sgRNAs for simultaneous disruption of T cell receptor (TCR) and human leukocyte antigen (HLA) molecules, supporting efficient generation of ‘universal’ CAR T cells.

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Fig. 1: Generation and evaluation of a minimal hU6 promoter devoid of nucleosome spacer sequence.
Fig. 2: Multiplex terminal CRISPR-CAR19 configuration with tandem CRISPR expression cassettes.
Fig. 3: Multiplex terminal configuration incorporating divergent scaffold sequences and minimal Pol III RNA promoters.
Fig. 4: Minimal multiplex Terminal configuration yields highly purified double HLA-TCRαβ- depleted population with CAR19+ enrichment.

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References

  1. White MD, Farmer M, Mirabile I, Brandner S, Collinge J, Mallucci GR. Single treatment with RNAi against prion protein rescues early neuronal dysfunction and prolongs survival in mice with prion disease. Proc Natl Acad Sci USA. 2008;105:10238–43.

    Article  CAS  Google Scholar 

  2. McBride JL, Boudreau RL, Harper SQ, Staber PD, Monteys AM, Martins I, et al. Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi. Proc Natl Acad Sci USA. 2008;105:5868–73.

    Article  CAS  Google Scholar 

  3. Boudreau RL, McBride JL, Martins I, Shen S, Xing Y, Carter BJ, et al. Nonallele-specific silencing of mutant and wild-type huntingtin demonstrates therapeutic efficacy in Huntington’s disease mice. Mol Ther. 2009;17:1053–63.

    Article  CAS  Google Scholar 

  4. Ren J, Liu X, Fang C, Jiang S, June CH, Zhao Y. Multiplex Genome Editing to Generate Universal CAR T Cells Resistant to PD1 Inhibition. Clin Cancer Res. 2017;23:2255–66.

    Article  CAS  Google Scholar 

  5. Georgiadis C, Preece R, Nickolay L, Etuk A, Petrova A, Ladon D, et al. Long Terminal repeat CRISPR-CAR-coupled “Universal” T cells mediate potent anti-leukemic effects. Mol Ther. 2018;26:1215–27.

    Article  CAS  Google Scholar 

  6. Gao Z, Herrera-Carrillo E, Berkhout B. RNA polymerase II activity of type 3 Pol III promoters. Mol Ther Nucleic Acids. 2018;12:135–45.

    Article  CAS  Google Scholar 

  7. Helbo AS, Lay FD, Jones PA, Liang G, Gronbaek K. Nucleosome positioning and NDR structure at RNA polymerase III promoters. Sci Rep. 2017;7:41947.

    Article  CAS  Google Scholar 

  8. Schramm L, Hernandez N. Recruitment of RNA polymerase III to its target promoters. Genes Dev. 2002;16:2593–620.

    Article  CAS  Google Scholar 

  9. Didychuk AL, Butcher SE, Brow DA. The life of U6 small nuclear RNA, from cradle to grave. RNA. 2018;24:437–60.

    Article  CAS  Google Scholar 

  10. Myslinski E, Ame JC, Krol A, Carbon P. An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene. Nucleic Acids Res. 2001;29:2502–9.

    Article  CAS  Google Scholar 

  11. Stunkel W, Kober I, Seifart KH. A nucleosome positioned in the distal promoter region activates transcription of the human U6 gene. Mol Cell Biol. 1997;17:4397–405.

    Article  CAS  Google Scholar 

  12. Zhao X, Pendergrast PS, Hernandez N. A positioned nucleosome on the human U6 promoter allows recruitment of SNAPc by the Oct-1 POU domain. Mol Cell. 2001;7:539–49.

    Article  CAS  Google Scholar 

  13. Murphy S, Yoon JB, Gerster T, Roeder RG. Oct-1 and Oct-2 potentiate functional interactions of a transcription factor with the proximal sequence element of small nuclear RNA genes. Mol Cell Biol. 1992;12:3247–61.

    Article  CAS  Google Scholar 

  14. Adamson B, Norman TM, Jost M, Cho MY, Nunez JK, Chen Y, et al. A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response. Cell. 2016;167:1867–82. e21.

    Article  CAS  Google Scholar 

  15. Houzet L, Jeang KT. MicroRNAs and human retroviruses. Biochim Biophys Acta. 2011;1809:686–93.

    Article  CAS  Google Scholar 

  16. Kao SY, Calman AF, Luciw PA, Peterlin BM. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature. 1987;330:489–93.

    Article  CAS  Google Scholar 

  17. Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM. Development of a self-inactivating lentivirus vector. J Virol. 1998;72:8150–7.

    Article  CAS  Google Scholar 

  18. Curtin JA, Dane AP, Swanson A, Alexander IE, Ginn SL. Bidirectional promoter interference between two widely used internal heterologous promoters in a late-generation lentiviral construct. Gene Ther. 2008;15:384–90.

    Article  CAS  Google Scholar 

  19. An W, Telesnitsky A. Frequency of direct repeat deletion in a human immunodeficiency virus type 1 vector during reverse transcription in human cells. Virology. 2001;286:475–82.

    Article  CAS  Google Scholar 

  20. Brake OT, Hooft K, Liu YP, Centlivre M, Jasmijn von Eije K, Berkhout B. Lentiviral vector design for multiple shRNA expression and durable HIV-1 inhibition. Mol Ther. 2008;16:557–64.

    Article  Google Scholar 

  21. Urbinati F, Arumugam P, Higashimoto T, Perumbeti A, Mitts K, Xia P, et al. Mechanism of reduction in titers from lentivirus vectors carrying large inserts in the 3’LTR. Mol Ther. 2009;17:1527–36.

    Article  CAS  Google Scholar 

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Acknowledgements

Supported by National Institute of health research (NIHR) and Great Ormond Street Biomedical Research Centre, (BRC) and Children with Cancer. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. Flow cytometry was performed at the UCL Great Ormond Street Institute of Child Health Flow Cytometry Core Facility, supported by the Great Ormond Street Children’s Charity (GOSHCC), grant reference U09822 (October 2007), and UCL Capital Equipment Funding, School of Life and Medical Sciences (September 2012).

Funding

This work was supported by the NIH Research (NIHR) (RP-2014-05-007), NIHR Blood and Transplant Research Units (BTRU) and Great Ormond Street Biomedical Research Centre (IS-BRC-1215-20012). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health.

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Author notes

  1. These authors contributed equally: Roland Preece, Christos Georgiadis

Authors and Affiliations

  1. Molecular and Cellular Immunology Unit, UCL Great Ormond Street Institute of Child Health, London, WC1N 1EH, UK

    Roland Preece, Christos Georgiadis, Soragia Athina Gkazi, Aniekan Etuk, Abraham Christi & Waseem Qasim

  2. NIHR Great Ormond Street Hospital Biomedical Research Centre, 30 Guilford Street, London, WC1N 1EH, UK

    Waseem Qasim

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  1. Roland Preece
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Correspondence to Christos Georgiadis.

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Conflict of interest

IP filed for Minimal U6 promoter N415912GB (RP, CG and WQ). WQ holds interests unrelated to this project in Autolus Ltd and Orchard Therapeutics. WQ received unrelated research funding from Cellectis, Servier, Miltenyi, Bellicum.

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Preece, R., Georgiadis, C., Gkazi, S.A. et al. ‘Mini’ U6 Pol III promoter exhibits nucleosome redundancy and supports multiplexed coupling of CRISPR/Cas9 effects. Gene Ther 27, 451–458 (2020). https://doi.org/10.1038/s41434-020-0142-z

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