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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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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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