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In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy.
J Gene Med. 2010 Apr; 12(4):354-64.JG

Abstract

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.

Authors+Show Affiliations

State Key Laboratory of AgroBiotech, The Life Science Research Centre, China Agricultural University, Beijing, China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

20235089

Citation

Wang, Qingsong, et al. "In Vitro Evaluation of Novel Antisense Oligonucleotides Is Predictive of in Vivo Exon Skipping Activity for Duchenne Muscular Dystrophy." The Journal of Gene Medicine, vol. 12, no. 4, 2010, pp. 354-64.
Wang Q, Yin H, Camelliti P, et al. In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy. J Gene Med. 2010;12(4):354-64.
Wang, Q., Yin, H., Camelliti, P., Betts, C., Moulton, H., Lee, H., Saleh, A. F., Gait, M. J., & Wood, M. J. (2010). In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy. The Journal of Gene Medicine, 12(4), 354-64. https://doi.org/10.1002/jgm.1446
Wang Q, et al. In Vitro Evaluation of Novel Antisense Oligonucleotides Is Predictive of in Vivo Exon Skipping Activity for Duchenne Muscular Dystrophy. J Gene Med. 2010;12(4):354-64. PubMed PMID: 20235089.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy. AU - Wang,Qingsong, AU - Yin,Haifang, AU - Camelliti,Patrizia, AU - Betts,Corinne, AU - Moulton,Hong, AU - Lee,Hyunil, AU - Saleh,Amer F, AU - Gait,Michael J, AU - Wood,Matthew J A, PY - 2010/3/18/entrez PY - 2010/3/18/pubmed PY - 2010/8/19/medline SP - 354 EP - 64 JF - The journal of gene medicine JO - J Gene Med VL - 12 IS - 4 N2 - 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. SN - 1521-2254 UR - https://www.unboundmedicine.com/medline/citation/20235089/In_vitro_evaluation_of_novel_antisense_oligonucleotides_is_predictive_of_in_vivo_exon_skipping_activity_for_Duchenne_muscular_dystrophy_ L2 - https://doi.org/10.1002/jgm.1446 DB - PRIME DP - Unbound Medicine ER -