Design and evaluation of locked nucleic acid-based splice-switching oligonucleotides in vitro.

Shimo T1, Tachibana K1, Saito K1, Yoshida T2, Tomita E3, Waki R1, Yamamoto T1, Doi T1, Inoue T2, Kawakami J4, Obika S5
Source: Nucleic Acids Res
Publication Date: (2014)
Issue: 42(12): 8174-87
Research Area:
Basic Research
Cells used in publication:
Skeletal Muscle Myoblast, (HSMM) human
Species: human
Tissue Origin: skeletal muscle
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. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.