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A Single Transcript Knockdown-Replacement Strategy Employing 5’ UTR Secondary Structures to Precisely Titrate Rescue Protein Translation

One overarching goal of gene therapy is the replacement of faulty genes with functional ones. A significant hurdle is presented by the fact that under- or over-expression of a protein may cause disease as readily as coding mutations. There is a clear and present need for pipelines to translate exper...

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Autores principales: Millette, Matthew M., Holland, Elizabeth D., Tenpas, Tanner J., Dent, Erik W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8995503/
https://www.ncbi.nlm.nih.gov/pubmed/35419562
http://dx.doi.org/10.3389/fgeed.2022.803375
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author Millette, Matthew M.
Holland, Elizabeth D.
Tenpas, Tanner J.
Dent, Erik W.
author_facet Millette, Matthew M.
Holland, Elizabeth D.
Tenpas, Tanner J.
Dent, Erik W.
author_sort Millette, Matthew M.
collection PubMed
description One overarching goal of gene therapy is the replacement of faulty genes with functional ones. A significant hurdle is presented by the fact that under- or over-expression of a protein may cause disease as readily as coding mutations. There is a clear and present need for pipelines to translate experimentally validated gene therapy strategies to clinical application. To address this we developed a modular, single-transgene expression system for replacing target genes with physiologically expressed variants. In order to accomplish this, we first designed a range of 5’ UTR “attenuator” sequences which predictably diminish translation of the paired gene. These sequences provide wide general utility by allowing control over translation from high expression, ubiquitous promoters. Importantly, we demonstrate that this permits an entirely novel knockdown and rescue application by pairing microRNA-adapted shRNAs alongside their respective replacement gene on a single transcript. A noteworthy candidate for this corrective approach is the degenerative and uniformly fatal motor neuron disease ALS. A strong proportion of non-idiopathic ALS cases are caused by varied mutations to the SOD1 gene, and as clinical trials to treat ALS are being initiated, it is important to consider that loss-of-function mechanisms contribute to its pathology as strongly as any other factor. As a generalized approach to treat monogenic diseases caused by heterogeneous mutations, we demonstrate complete and predictable control over replacement of SOD1 in stable cell lines by varying the strength of attenuators.
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spelling pubmed-89955032022-04-12 A Single Transcript Knockdown-Replacement Strategy Employing 5’ UTR Secondary Structures to Precisely Titrate Rescue Protein Translation Millette, Matthew M. Holland, Elizabeth D. Tenpas, Tanner J. Dent, Erik W. Front Genome Ed Genome Editing One overarching goal of gene therapy is the replacement of faulty genes with functional ones. A significant hurdle is presented by the fact that under- or over-expression of a protein may cause disease as readily as coding mutations. There is a clear and present need for pipelines to translate experimentally validated gene therapy strategies to clinical application. To address this we developed a modular, single-transgene expression system for replacing target genes with physiologically expressed variants. In order to accomplish this, we first designed a range of 5’ UTR “attenuator” sequences which predictably diminish translation of the paired gene. These sequences provide wide general utility by allowing control over translation from high expression, ubiquitous promoters. Importantly, we demonstrate that this permits an entirely novel knockdown and rescue application by pairing microRNA-adapted shRNAs alongside their respective replacement gene on a single transcript. A noteworthy candidate for this corrective approach is the degenerative and uniformly fatal motor neuron disease ALS. A strong proportion of non-idiopathic ALS cases are caused by varied mutations to the SOD1 gene, and as clinical trials to treat ALS are being initiated, it is important to consider that loss-of-function mechanisms contribute to its pathology as strongly as any other factor. As a generalized approach to treat monogenic diseases caused by heterogeneous mutations, we demonstrate complete and predictable control over replacement of SOD1 in stable cell lines by varying the strength of attenuators. Frontiers Media S.A. 2022-03-28 /pmc/articles/PMC8995503/ /pubmed/35419562 http://dx.doi.org/10.3389/fgeed.2022.803375 Text en Copyright © 2022 Millette, Holland, Tenpas and Dent. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Genome Editing
Millette, Matthew M.
Holland, Elizabeth D.
Tenpas, Tanner J.
Dent, Erik W.
A Single Transcript Knockdown-Replacement Strategy Employing 5’ UTR Secondary Structures to Precisely Titrate Rescue Protein Translation
title A Single Transcript Knockdown-Replacement Strategy Employing 5’ UTR Secondary Structures to Precisely Titrate Rescue Protein Translation
title_full A Single Transcript Knockdown-Replacement Strategy Employing 5’ UTR Secondary Structures to Precisely Titrate Rescue Protein Translation
title_fullStr A Single Transcript Knockdown-Replacement Strategy Employing 5’ UTR Secondary Structures to Precisely Titrate Rescue Protein Translation
title_full_unstemmed A Single Transcript Knockdown-Replacement Strategy Employing 5’ UTR Secondary Structures to Precisely Titrate Rescue Protein Translation
title_short A Single Transcript Knockdown-Replacement Strategy Employing 5’ UTR Secondary Structures to Precisely Titrate Rescue Protein Translation
title_sort single transcript knockdown-replacement strategy employing 5’ utr secondary structures to precisely titrate rescue protein translation
topic Genome Editing
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8995503/
https://www.ncbi.nlm.nih.gov/pubmed/35419562
http://dx.doi.org/10.3389/fgeed.2022.803375
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