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Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy.
J Gene Med. 2002 Nov-Dec; 4(6):644-54.JG

Abstract

BACKGROUND

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by dystrophin gene mutations that preclude synthesis of a functional protein. One potential treatment of the disorder has utilised antisense oligoribonucleotides (AOs) to induce removal of disease-associated exons during pre-mRNA processing. Induced in-frame mRNA transcripts encode a shorter but functional dystrophin. We have investigated and improved the design of AOs capable of removing exon 23, and thus the disease-causing nonsense mutation, from mRNA in the mdx mouse model of DMD.

METHODS

H-2K(b)-tsA58 mdx cultures were transfected with complexes of Lipofectin and AOs. Exon skipping was detected by RT-PCR and subsequent protein production was demonstrated by Western blotting. AOs were delivered at a range of doses in order to compare relative efficiencies.

RESULTS

We describe effective and reproducible exon 23 skipping with several AOs, including one as small as 17 nucleotides. Furthermore, the location of a sensitive exon 23 target site has been refined, whilst minimum effective doses have been estimated in vitro. These doses are significantly lower than previously reported and were associated with the synthesis of dystrophin protein in vitro.

CONCLUSIONS

These results demonstrate the increasing feasibility of an AO-based therapy for treatment of DMD. By refining AO design we have been able to reduce the size and the effective dose of the AOs and have dramatically improved the efficiency of the technique.

Authors+Show Affiliations

Australian Neuromuscular Research Institute, Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Perth, Western Australia, 6907.No 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

12439856

Citation

Mann, Christopher J., et al. "Improved Antisense Oligonucleotide Induced Exon Skipping in the Mdx Mouse Model of Muscular Dystrophy." The Journal of Gene Medicine, vol. 4, no. 6, 2002, pp. 644-54.
Mann CJ, Honeyman K, McClorey G, et al. Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy. J Gene Med. 2002;4(6):644-54.
Mann, C. J., Honeyman, K., McClorey, G., Fletcher, S., & Wilton, S. D. (2002). Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy. The Journal of Gene Medicine, 4(6), 644-54.
Mann CJ, et al. Improved Antisense Oligonucleotide Induced Exon Skipping in the Mdx Mouse Model of Muscular Dystrophy. J Gene Med. 2002 Nov-Dec;4(6):644-54. PubMed PMID: 12439856.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy. AU - Mann,Christopher J, AU - Honeyman,Kaite, AU - McClorey,Graham, AU - Fletcher,Sue, AU - Wilton,Stephen D, PY - 2002/11/20/pubmed PY - 2003/5/29/medline PY - 2002/11/20/entrez SP - 644 EP - 54 JF - The journal of gene medicine JO - J Gene Med VL - 4 IS - 6 N2 - BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by dystrophin gene mutations that preclude synthesis of a functional protein. One potential treatment of the disorder has utilised antisense oligoribonucleotides (AOs) to induce removal of disease-associated exons during pre-mRNA processing. Induced in-frame mRNA transcripts encode a shorter but functional dystrophin. We have investigated and improved the design of AOs capable of removing exon 23, and thus the disease-causing nonsense mutation, from mRNA in the mdx mouse model of DMD. METHODS: H-2K(b)-tsA58 mdx cultures were transfected with complexes of Lipofectin and AOs. Exon skipping was detected by RT-PCR and subsequent protein production was demonstrated by Western blotting. AOs were delivered at a range of doses in order to compare relative efficiencies. RESULTS: We describe effective and reproducible exon 23 skipping with several AOs, including one as small as 17 nucleotides. Furthermore, the location of a sensitive exon 23 target site has been refined, whilst minimum effective doses have been estimated in vitro. These doses are significantly lower than previously reported and were associated with the synthesis of dystrophin protein in vitro. CONCLUSIONS: These results demonstrate the increasing feasibility of an AO-based therapy for treatment of DMD. By refining AO design we have been able to reduce the size and the effective dose of the AOs and have dramatically improved the efficiency of the technique. SN - 1099-498X UR - https://www.unboundmedicine.com/medline/citation/12439856/Improved_antisense_oligonucleotide_induced_exon_skipping_in_the_mdx_mouse_model_of_muscular_dystrophy_ L2 - https://doi.org/10.1002/jgm.295 DB - PRIME DP - Unbound Medicine ER -