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A compact and simple method of achieving differential transgene expression by exploiting translational readthrough

Sillibourne, James E; Agliardi, Giulia; Righi, Matteo; Smetanova, Katerina; Rowley, Grant; Speller, Simon; Dolor, Abigail; ... Pule, Martin; + view all (2022) A compact and simple method of achieving differential transgene expression by exploiting translational readthrough. Biotechniques , 72 (4) 10.2144/btn-2021-0079. (In press). Green open access

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Abstract

The development of multicistronic vectors enabling differential transgene expression is a goal of gene therapy and poses a significant engineering challenge. Current approaches rely on the insertion of long regulatory sequences that occupy valuable space in vectors, which have a finite and limited packaging capacity. Here we describe a simple method of achieving differential transgene expression by inserting stop codons and translational readthrough motifs (TRMs) to suppress stop codon termination. TRMs reduced downstream transgene expression ∼sixfold to ∼140-fold, depending on the combination of stop codon and TRM used. We show that a TRM can facilitate the controlled secretion of the highly potent cytokine IL-12 at therapeutically beneficial levels in an aggressive immunocompetent mouse melanoma model to prevent tumor growth. Given their compact size (6 bp) and ease of introduction, we envisage that TRMs will be widely adopted in recombinant DNA engineering to facilitate differential transgene expression.

Type: Article
Title: A compact and simple method of achieving differential transgene expression by exploiting translational readthrough
Location: England
Open access status: An open access version is available from UCL Discovery
DOI: 10.2144/btn-2021-0079
Publisher version: https://doi.org/10.2144/btn-2021-0079
Language: English
Additional information: This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/
Keywords: T cell, cancer, chimeric antigen receptor, differential transgene expression, immunotherapy, packaging limit, translational readthrough motif, ultra-low expression
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute > Research Department of Haematology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute
URI: https://discovery.ucl.ac.uk/id/eprint/10144895
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