In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy.
BACKGROUND
Targeted splice modulation of pre-mRNA transcripts by antisense oligonucleotides (AOs) can correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) arises as a result of mutations that interrupt the open-reading frame in the DMD gene encoding dystrophin such that dystrophin protein is absent, leading to fatal muscle degeneration. AOs have been shown to correct this dystrophin defect via exon skipping to yield functional dystrophin protein in animal models of DMD and also in DMD patients via intramuscular administration. To advance this therapeutic method requires increased exon skipping efficiency via an optimized AO sequence, backbone chemistry and additional modifications, and the improvement of methods for evaluating AO efficacy.


METHODS
In the present study, we establish the conditions for rapid in vitro AO screening in H(2)K muscle cells, in which we evaluate the exon skipping properties of a number of known and novel AO chemistries [2'-O-methyl, peptide nucleic acid, phosphorodiamidate morpholino (PMO)] and their peptide-conjugated derivatives and correlate their in vitro and in vivo exon skipping activities.


RESULTS
The present study demonstrates that using AO concentrations of 300 nM with analysis at a single time-point of 24 h post-transfection allowed the effective in vitro screening of AO compounds to yield data predictive of in vivo exon skipping efficacy. Peptide-conjugated PMO AOs provided the highest in vitro activity. We also show for the first time that the feasibility of rapid AO screening extends to primary cardiomyocytes.


CONCLUSIONS
In vitro screening of different AOs within the same chemical class is a reliable method for predicting the in vivo exon skipping efficiency of AOs for DMD.
Wang
Qingsong
Q
State Key Laboratory of AgroBiotech, The Life Science Research Centre, China Agricultural University, Beijing, China.
Yin
Haifang
H
Camelliti
Patrizia
P
Betts
Corinne
C
Moulton
Hong
H
Lee
Hyunil
H
Saleh
Amer F
AF
Gait
Michael J
MJ
Wood
Matthew J A
MJ
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D016189:Dystrophin / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D051379:Mice; D018101:Mice, Inbred mdx; D008969:Molecular Sequence Data; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000628:therapy*; D032383:Myocytes, Cardiac; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection
D017931:DNA Primers; D016189:Dystrophin; D016376:Oligonucleotides, Antisense; D012322:RNA Precursors