Oligonucleotide comprising an inosine for treating dmd

a technology of inosine and oligonucleotide, which is applied in the field of molecular biology and medicine, can solve the problems of calcium-activated proteases and fiber necrosis, progressive muscle wasting and weakness, and/or abnormal formation of dystrophins

Inactive Publication Date: 2012-02-23
BIOMARIN TECH BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In Duchenne dystrophin is absent, whereas in Becker some dystrophin is present but its production is most often not sufficient and/or the dystrophin present is abnormally formed.
The frameshift in the DMD gene's transcript (mRNA) results in the production of a truncated non-functional dystrophin protein, resulting in progressive muscle wasting and weakness.
In the absence of dystrophin, mechanical stress leads to sarcolemmal ru

Method used

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  • Oligonucleotide comprising an inosine for treating dmd
  • Oligonucleotide comprising an inosine for treating dmd

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example 1

Materials and Methods

[0155]AON design was based on (partly) overlapping open secondary structures of the target exon RNA as predicted by the m-fold program, on (partly) overlapping putative SR-protein binding sites as predicted by the ESE-finder software. AONs were synthesized by Prosensa Therapeutics B.V. (Leiden, Netherlands), and contain 2′-O-methyl RNA and full-length phosphorothioate (PS) backbones.

Tissue Culturing, Transfection and RT-PCR Analysis

[0156]Myotube cultures derived from a healthy individual (“human control”) (examples 1, 3, and 4; exon 43, 50, 52 skipping) or a DMD patient carrying an exon 45 deletion (example 2; exon 46 skipping) were processed as described previously (Aartsma-Rus et al., Neuromuscul. Disord. 2002; 12: S71-77 and Hum Mol Genet. 2003; 12(8): 907-14). For the screening of AONs, myotube cultures were transfected with 200 nM for each AON (PS220 and PS305). Transfection reagent UNIFectylin (Prosensa Therapeutics BV, Netherlands) was used, with 2 μl UNI...

example 2

[0159]Materials and Methods

[0160]AON design was based on (partly) overlapping open secondary structures of the target exon 45 RNA as predicted by the m-fold program, on (partly) overlapping putative SR-protein binding sites as predicted by the ESE-finder software. AONs were synthesized by Prosensa Therapeutics B.V. (Leiden, Netherlands), and contain 2′-O-methyl RNA, full-length phosphorothioate (PS) backbones and one inosine for guanosine substitution.

Tissue Culturing, Transfection and RT-PCR Analysis

[0161]Myotube cultures derived from a healthy individual (“human control”) were processed as described previously (Aartsma-Rus et al., Neuromuscul. Disord. 2002; 12: S71-77 and Hum Mol Genet. 2003; 12(8): 907-14). For the screening of AONs, myotube cultures were transfected with 200 nM for each AON. Transfection reagent UNIFectylin (Prosensa Therapeutics BV, Netherlands) was used, with 2 μl UNIFectylin per μg AON. Exon skipping efficiencies were determined by nested RT-PCR analysis usin...

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Abstract

The invention provides an oligonucleotide comprising an inosine, and/or a nucleotide containing a base able to form a wobble base pair or a functional equivalent thereof, wherein the oligonucleotide, or a functional equivalent thereof, comprises a sequence which is complementary to at least part of a dystrophin pre-m RNA exon or at least part of a non-exon region of a dystrophin pre-m RNA said part being a contiguous stretch comprising at least 8 nucleotides. The invention further provides the use of said oligonucleotide for preventing or treating DMD or BMD.

Description

FIELD OF THE INVENTION[0001]The invention relates to the fields of molecular biology and medicine.BACKGROUND OF THE INVENTION[0002]A muscle disorder is a disease that usually has a significant impact on the life of an individual. A muscle disorder can have either a genetic cause or a non-genetic cause. An important group of muscle diseases with a genetic cause are Becker Muscular Dystrophy (BMD) and Duchenne Muscular Dystrophy (DMD). These disorders are caused by defects in a gene for a muscle protein.[0003]Becker Muscular Dystrophy and Duchenne Muscular Dystrophy are genetic muscular dystrophies with a relatively high incidence. In both Duchenne and Becker muscular dystrophy the muscle protein dystrophin is affected. In Duchenne dystrophin is absent, whereas in Becker some dystrophin is present but its production is most often not sufficient and / or the dystrophin present is abnormally formed. Both diseases are associated with recessive X-linked inheritance. DMD results from a frame...

Claims

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Application Information

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IPC IPC(8): A61K31/7088C07H21/04A61P29/00A61K31/7125A61P21/00C07H21/02A61K31/712
CPCC12N15/111C12N15/113C12N2310/11C12N2310/315C12N2310/321C12N2320/33C12N2310/331C12N2310/3521A61P21/00A61P29/00A61K48/00C12N2310/333C12N2310/3231C12N2310/3181C12N2310/336
Inventor VAN DEUTEKOM, JUDITH CHRISTINA THEODORADE KIMPE, JOSEPHUS JOHANNESPLATENBURG, GERARDUS JOHANNES
Owner BIOMARIN TECH BV
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