Johari, Y.B., Mercer, A.C., Liu, Y. et al. (2 more authors) (2021) Design of synthetic promoters for controlled expression of therapeutic genes in retinal pigment epithelial cells. Biotechnology and Bioengineering, 118 (5). pp. 2001-2015. ISSN 0006-3592
Abstract
Age‐related macular degeneration (AMD) associated with dysfunction of retinal pigment epithelial (RPE) cells is the most common cause of untreatable blindness. To advance gene therapy as a viable treatment for AMD there is a need for technologies that enable controlled, RPE‐specific expression of therapeutic genes. Here we describe design, construction and testing of compact synthetic promoters with a pre‐defined transcriptional activity and RPE cell specificity. Initial comparative informatic analyses of RPE and photoreceptor (PR) cell transcriptomic data identified conserved and overrepresented transcription factor regulatory elements (TFREs, 8–19 bp) specifically associated with transcriptionally active RPE genes. Both RPE‐specific TFREs and those derived from the generically active cytomegalovirus‐immediate early (CMV‐IE) promoter were then screened in vitro to identify sequence elements able to control recombinant gene transcription in model induced pluripotent stem (iPS)‐derived and primary human RPE cells. Two libraries of heterotypic synthetic promoters varying in predicted RPE specificity and transcriptional activity were designed de novo using combinations of up to 20 discrete TFREs in series (323–602 bp) and their transcriptional activity in model RPE cells was compared to that of the endogenous BEST1 promoter (661 bp, plus an engineered derivative) and the highly active generic CMV‐IE promoter (650 bp). Synthetic promoters with a highpredicted specificity, comprised predominantly of endogenous TFREs exhibited a range of activities up to 8‐fold that of the RPE‐specific BEST1 gene promoter. Moreover, albeit at a lower predicted specificity, synthetic promoter transcriptional activity in model RPE cells was enhanced beyond that of the CMV‐IE promoter when viral elements were utilized in combination with endogenous RPE‐specific TFREs, with a reduction in promoter size of 15%. Taken together, while our data reveal an inverse relationship between synthetic promoter activity and cell‐type specificity, cell context‐specific control of recombinant gene transcriptional activity may be achievable.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2021 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals LLC. This is an open access article under the terms of the Creative Commons Attribution License, (http://creativecommons.org/licenses/by/4.0/) which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
Keywords: | cell‐specific expression; gene therapy; RPE cells; synthetic biology; synthetic promoter; transcriptomics |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 15 Mar 2021 13:43 |
Last Modified: | 15 Feb 2022 12:37 |
Status: | Published |
Publisher: | Wiley |
Refereed: | Yes |
Identification Number: | 10.1002/bit.27713 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:171769 |