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Optimizing RNA/ENA chimeric antisense oligonucleotides using in vitro splicing.
Methods Mol Biol. 2012; 867:131-41.MM

Abstract

A molecular therapy for Duchenne muscular dystrophy (DMD) that converts dystrophin mRNA from out-of-frame to in-frame transcripts by inducing exon skipping with antisense oligonucleotides (AOs) is now approaching clinical application. To exploit the broad therapeutic applicability of exon skipping therapy, it is necessary to identify AOs that are able to induce efficient and specific exon skipping. To optimize AOs, we have established an in vitro splicing system using cultured DMD myocytes. Here, we describe the process of identifying the best AO.Cultured DMD myocytes are established from a biopsy sample and the target exon is chosen. A series of AOs are designed to cover the whole target exon sequence. As AOs, we use 15-20-mer chimeric oligonucleotides consisting of 2'-O-methyl RNA and modified nucleic acid (2'-O, 4'-C-ethylene-bridged nucleic acid). Each AO is transfected individually into cultured DMD myocytes, and the resulting mRNA is analyzed by reverse transcription-PCR. The ability of each AO to induce exon skipping is examined by comparing the amount of cDNA with and without exon skipping. If necessary, having roughly localized the target region, another set of AOs are designed and the exon skipping abilities of the new AOs are examined. Finally, one AO is determined as the best for the molecular therapy.Our simple and reliable methods using an in vitro splicing system have enabled us to identify optimized AOs against many exons of the DMD gene.

Authors+Show Affiliations

Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan. takesima@med.kobe-u.ac.jpNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

22454059

Citation

Takeshima, Yasuhiro, et al. "Optimizing RNA/ENA Chimeric Antisense Oligonucleotides Using in Vitro Splicing." Methods in Molecular Biology (Clifton, N.J.), vol. 867, 2012, pp. 131-41.
Takeshima Y, Yagi M, Matsuo M. Optimizing RNA/ENA chimeric antisense oligonucleotides using in vitro splicing. Methods Mol Biol. 2012;867:131-41.
Takeshima, Y., Yagi, M., & Matsuo, M. (2012). Optimizing RNA/ENA chimeric antisense oligonucleotides using in vitro splicing. Methods in Molecular Biology (Clifton, N.J.), 867, 131-41. https://doi.org/10.1007/978-1-61779-767-5_9
Takeshima Y, Yagi M, Matsuo M. Optimizing RNA/ENA Chimeric Antisense Oligonucleotides Using in Vitro Splicing. Methods Mol Biol. 2012;867:131-41. PubMed PMID: 22454059.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Optimizing RNA/ENA chimeric antisense oligonucleotides using in vitro splicing. AU - Takeshima,Yasuhiro, AU - Yagi,Mariko, AU - Matsuo,Masafumi, PY - 2012/3/29/entrez PY - 2012/3/29/pubmed PY - 2012/7/24/medline SP - 131 EP - 41 JF - Methods in molecular biology (Clifton, N.J.) JO - Methods Mol Biol VL - 867 N2 - A molecular therapy for Duchenne muscular dystrophy (DMD) that converts dystrophin mRNA from out-of-frame to in-frame transcripts by inducing exon skipping with antisense oligonucleotides (AOs) is now approaching clinical application. To exploit the broad therapeutic applicability of exon skipping therapy, it is necessary to identify AOs that are able to induce efficient and specific exon skipping. To optimize AOs, we have established an in vitro splicing system using cultured DMD myocytes. Here, we describe the process of identifying the best AO.Cultured DMD myocytes are established from a biopsy sample and the target exon is chosen. A series of AOs are designed to cover the whole target exon sequence. As AOs, we use 15-20-mer chimeric oligonucleotides consisting of 2'-O-methyl RNA and modified nucleic acid (2'-O, 4'-C-ethylene-bridged nucleic acid). Each AO is transfected individually into cultured DMD myocytes, and the resulting mRNA is analyzed by reverse transcription-PCR. The ability of each AO to induce exon skipping is examined by comparing the amount of cDNA with and without exon skipping. If necessary, having roughly localized the target region, another set of AOs are designed and the exon skipping abilities of the new AOs are examined. Finally, one AO is determined as the best for the molecular therapy.Our simple and reliable methods using an in vitro splicing system have enabled us to identify optimized AOs against many exons of the DMD gene. SN - 1940-6029 UR - https://www.unboundmedicine.com/medline/citation/22454059/Optimizing_RNA/ENA_chimeric_antisense_oligonucleotides_using_in_vitro_splicing_ L2 - https://dx.doi.org/10.1007/978-1-61779-767-5_9 DB - PRIME DP - Unbound Medicine ER -