Allele-Specific Prevention of Nonsense-Mediated Decay in Cystic Fibrosis Using Homology-Independent Genome Editing
Nonsense-mediated decay (NMD) is a major pathogenic mechanism underlying a diversity of genetic disorders. Nonsense variants tend to lead to more severe disease phenotypes and are often difficult targets for small molecule therapeutic development as a result of insufficient protein production. The t...
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doaj-eb2405ef414a48d59db42b474b399baf2020-11-25T02:39:55ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012020-06-011711181128Allele-Specific Prevention of Nonsense-Mediated Decay in Cystic Fibrosis Using Homology-Independent Genome EditingSteven Erwood0Onofrio Laselva1Teija M.I. Bily2Reid A. Brewer3Alexandra H. Rutherford4Christine E. Bear5Evgueni A. Ivakine6Program in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, CanadaProgram in Molecular Medicine, The Hospital for Sick Children Research Institute, Toronto, ON, Canada; Department of Physiology, University of Toronto, Toronto, ON, CanadaProgram in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, ON, CanadaProgram in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, ON, CanadaProgram in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, ON, CanadaProgram in Molecular Medicine, The Hospital for Sick Children Research Institute, Toronto, ON, Canada; Department of Physiology, University of Toronto, Toronto, ON, Canada; Department of Biochemistry, University of Toronto, Toronto, ON, CanadaProgram in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, ON, Canada; Department of Physiology, University of Toronto, Toronto, ON, Canada; Corresponding author: Evgueni A. Ivakine, Program in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, ON, Canada.Nonsense-mediated decay (NMD) is a major pathogenic mechanism underlying a diversity of genetic disorders. Nonsense variants tend to lead to more severe disease phenotypes and are often difficult targets for small molecule therapeutic development as a result of insufficient protein production. The treatment of cystic fibrosis (CF), an autosomal recessive disease caused by mutations in the CFTR gene, exemplifies the challenge of therapeutically addressing nonsense mutations in human disease. Therapeutic development in CF has led to multiple, highly successful protein modulatory interventions, yet no targeted therapies have been approved for nonsense mutations. Here, we have designed a CRISPR-Cas9-based strategy for the targeted prevention of NMD of CFTR transcripts containing the second most common nonsense variant listed in CFTR2, W1282X. By introducing a deletion of the downstream genic region following the premature stop codon, we demonstrate significantly increased protein expression of this mutant variant. Notably, in combination with protein modulators, genome editing significantly increases the potentiated channel activity of W1282X-CFTR in human bronchial epithelial cells. Furthermore, we show how the outlined approach can be modified to permit allele-specific editing. The described approach can be extended to other late-occurring nonsense mutations in the CFTR gene or applied as a generalized approach for gene-specific prevention of NMD in disorders where a truncated protein product retains full or partial functionality.http://www.sciencedirect.com/science/article/pii/S2329050120300899genome editingallele-specificityTRIKAFTAcystic fibrosisCRISPR-Cas9nonsense-mediated decay |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Steven Erwood Onofrio Laselva Teija M.I. Bily Reid A. Brewer Alexandra H. Rutherford Christine E. Bear Evgueni A. Ivakine |
spellingShingle |
Steven Erwood Onofrio Laselva Teija M.I. Bily Reid A. Brewer Alexandra H. Rutherford Christine E. Bear Evgueni A. Ivakine Allele-Specific Prevention of Nonsense-Mediated Decay in Cystic Fibrosis Using Homology-Independent Genome Editing Molecular Therapy: Methods & Clinical Development genome editing allele-specificity TRIKAFTA cystic fibrosis CRISPR-Cas9 nonsense-mediated decay |
author_facet |
Steven Erwood Onofrio Laselva Teija M.I. Bily Reid A. Brewer Alexandra H. Rutherford Christine E. Bear Evgueni A. Ivakine |
author_sort |
Steven Erwood |
title |
Allele-Specific Prevention of Nonsense-Mediated Decay in Cystic Fibrosis Using Homology-Independent Genome Editing |
title_short |
Allele-Specific Prevention of Nonsense-Mediated Decay in Cystic Fibrosis Using Homology-Independent Genome Editing |
title_full |
Allele-Specific Prevention of Nonsense-Mediated Decay in Cystic Fibrosis Using Homology-Independent Genome Editing |
title_fullStr |
Allele-Specific Prevention of Nonsense-Mediated Decay in Cystic Fibrosis Using Homology-Independent Genome Editing |
title_full_unstemmed |
Allele-Specific Prevention of Nonsense-Mediated Decay in Cystic Fibrosis Using Homology-Independent Genome Editing |
title_sort |
allele-specific prevention of nonsense-mediated decay in cystic fibrosis using homology-independent genome editing |
publisher |
Elsevier |
series |
Molecular Therapy: Methods & Clinical Development |
issn |
2329-0501 |
publishDate |
2020-06-01 |
description |
Nonsense-mediated decay (NMD) is a major pathogenic mechanism underlying a diversity of genetic disorders. Nonsense variants tend to lead to more severe disease phenotypes and are often difficult targets for small molecule therapeutic development as a result of insufficient protein production. The treatment of cystic fibrosis (CF), an autosomal recessive disease caused by mutations in the CFTR gene, exemplifies the challenge of therapeutically addressing nonsense mutations in human disease. Therapeutic development in CF has led to multiple, highly successful protein modulatory interventions, yet no targeted therapies have been approved for nonsense mutations. Here, we have designed a CRISPR-Cas9-based strategy for the targeted prevention of NMD of CFTR transcripts containing the second most common nonsense variant listed in CFTR2, W1282X. By introducing a deletion of the downstream genic region following the premature stop codon, we demonstrate significantly increased protein expression of this mutant variant. Notably, in combination with protein modulators, genome editing significantly increases the potentiated channel activity of W1282X-CFTR in human bronchial epithelial cells. Furthermore, we show how the outlined approach can be modified to permit allele-specific editing. The described approach can be extended to other late-occurring nonsense mutations in the CFTR gene or applied as a generalized approach for gene-specific prevention of NMD in disorders where a truncated protein product retains full or partial functionality. |
topic |
genome editing allele-specificity TRIKAFTA cystic fibrosis CRISPR-Cas9 nonsense-mediated decay |
url |
http://www.sciencedirect.com/science/article/pii/S2329050120300899 |
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