title: Design and evaluation of locked nucleic acid-based splice-switching oligonucleotides in vitro.
abstract: Antisense-mediated modulation of pre-mRNA splicing is an attractive therapeutic strategy for genetic diseases. Currently, there are few examples of modulation of pre-mRNA splicing using locked nucleic acid (LNA) antisense oligonucleotides, and, in particular, no systematic study has addressed the optimal design of LNA-based splice-switching oligonucleotides (LNA SSOs). Here, we designed a series of LNA SSOs complementary to the human dystrophin exon 58 sequence and evaluated their ability to induce exon skipping in vitro using reverse transcription-polymerase chain reaction. We demonstrated that the number of LNAs in the SSO sequence and the melting temperature of the SSOs play important roles in inducing exon skipping and seem to be key factors for designing efficient LNA SSOs. LNA SSO length was an important determinant of activity: a 13-mer with six LNA modifications had the highest efficacy, and a 7-mer was the minimal length required to induce exon skipping. Evaluation of exon skipping activity using mismatched LNA/DNA mixmers revealed that 9-mer LNA SSO allowed a better mismatch discrimination. LNA SSOs also induced exon skipping of endogenous human dystrophin in primary human skeletal muscle cells. Taken together, our findings indicate that LNA SSOs are powerful tools for modulating pre-mRNA splicing.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Takenori
authors.initials: T
authors.lastname: Shimo
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Tachibana
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Kiwamu
authors.initials: K
authors.lastname: Saito
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan Division of Cellular and Gene Therapy Products, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors.forename: Tokuyuki
authors.initials: T
authors.lastname: Yoshida
authors.affiliation: Department of Nanobiochemistry, FIRST, Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.
authors.forename: Erisa
authors.initials: E
authors.lastname: Tomita
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Waki
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Takefumi
authors.initials: T
authors.lastname: Doi
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan Division of Cellular and Gene Therapy Products, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Inoue
authors.affiliation: Department of Nanobiochemistry, FIRST, Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Kawakami
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan obika@phs.osaka-u.ac.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Obika
mesh_terms: D000293:Adolescent; D020137:Base Pair Mismatch; D002460:Cell Line; D002478:Cells, Cultured; D016189:Dystrophin / Q000235:genetics; D005091:Exons; D005260:Female; D006801:Humans; D018482:Muscle, Skeletal / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry*; D012326:RNA Splicing
chemical_list: D016189:Dystrophin; D009841:Oligonucleotides; C477371:locked nucleic acid