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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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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

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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  2. Christos Georgiadis
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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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  • DOI: https://doi.org/10.1038/s41434-020-0142-z

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