UZH-Logo

Maintenance Infos

A sensitive assay system to test antisense oligonucleotides for splice suppression therapy in the mouse liver


Gallego-Villar, Lorena; Viecelli, Hiu Man; Pérez, Belén; Harding, Cary O; Ugarte, Magdalena; Thöny, Beat; Desviat, Lourdes R (2014). A sensitive assay system to test antisense oligonucleotides for splice suppression therapy in the mouse liver. Molecular Therapy - Nucleic Acids, 3:e193.

Abstract

We have previously demonstrated the efficacy of antisense therapy for splicing defects in cellular models of metabolic diseases, suppressing the use of cryptic splice sites or pseudoexon insertions. To date, no animal models with these defects are available. Here, we propose exon skipping of the phenylalanine hydroxylase (Pah) gene expressed in liver and kidney to generate systemic hyperphenylalaninemia in mice as a sensitive in vivo assay to test splice suppression. Systemic elevation of blood L-Phe can be quantified using tandem MS/MS. Exon 11 and/or 12 skipping for the normal PAH gene was validated in hepatoma cells for comparing two oligonucleotide chemistries, morpholinos and locked nucleic acids. Subsequently, Vivo-morpholinos (VMO) were tested in wild-type and in phenotypically normal Pah(enu2/+) heterozygous mice to target exon 11 and/or 12 of the murine Pah gene using different VMO dosing, mode of injection and treatment regimes. Consecutive intravenous injections of VMO resulted in transient hyperphenylalaninemia correlating with complete exon skipping and absence of PAH protein and enzyme activity. Sustained effect required repeated injection of VMOs. Our results provide not only a sensitive in vivo assay to test for splice-modulating antisense oligonucleotides, but also a simple method to generate murine models for genetic liver diseases.

We have previously demonstrated the efficacy of antisense therapy for splicing defects in cellular models of metabolic diseases, suppressing the use of cryptic splice sites or pseudoexon insertions. To date, no animal models with these defects are available. Here, we propose exon skipping of the phenylalanine hydroxylase (Pah) gene expressed in liver and kidney to generate systemic hyperphenylalaninemia in mice as a sensitive in vivo assay to test splice suppression. Systemic elevation of blood L-Phe can be quantified using tandem MS/MS. Exon 11 and/or 12 skipping for the normal PAH gene was validated in hepatoma cells for comparing two oligonucleotide chemistries, morpholinos and locked nucleic acids. Subsequently, Vivo-morpholinos (VMO) were tested in wild-type and in phenotypically normal Pah(enu2/+) heterozygous mice to target exon 11 and/or 12 of the murine Pah gene using different VMO dosing, mode of injection and treatment regimes. Consecutive intravenous injections of VMO resulted in transient hyperphenylalaninemia correlating with complete exon skipping and absence of PAH protein and enzyme activity. Sustained effect required repeated injection of VMOs. Our results provide not only a sensitive in vivo assay to test for splice-modulating antisense oligonucleotides, but also a simple method to generate murine models for genetic liver diseases.

Citations

1 citation in Web of Science®
1 citation in Scopus®
Google Scholar™

Altmetrics

Downloads

22 downloads since deposited on 13 Feb 2015
20 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Children's Hospital Zurich > Medical Clinic
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2014
Deposited On:13 Feb 2015 15:51
Last Modified:05 Apr 2016 18:42
Publisher:Nature Publishing Group
ISSN:2162-2531
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1038/mtna.2014.44
PubMed ID:25226162
Permanent URL: https://doi.org/10.5167/uzh-103324

Download

[img]
Preview
Content: Published Version
Filetype: PDF
Size: 1MB
View at publisher

TrendTerms

TrendTerms displays relevant terms of the abstract of this publication and related documents on a map. The terms and their relations were extracted from ZORA using word statistics. Their timelines are taken from ZORA as well. The bubble size of a term is proportional to the number of documents where the term occurs. Red, orange, yellow and green colors are used for terms that occur in the current document; red indicates high interlinkedness of a term with other terms, orange, yellow and green decreasing interlinkedness. Blue is used for terms that have a relation with the terms in this document, but occur in other documents.
You can navigate and zoom the map. Mouse-hovering a term displays its timeline, clicking it yields the associated documents.

Author Collaborations