Tricyclo-dna antisense oligonucleotides, compositions, and methods for the treatment of disease

a technology of antisense oligonucleotides and tricyclodna, applied in the field of synthetic antisense oligonucleotides (aon), can solve the problems of inability to produce dystrophin in striated muscles, affecting the function of striated muscles, and affecting the function of muscle fibers, etc., and achieve the effect of finding utility

Inactive Publication Date: 2012-06-14
INST NAT DE LA SANTE & DE LA RECHERCHE MEDICALE (INSERM) +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0049]As described in greater detail, below, exon skipping is achieved in the nucleus during the maturation process of pre-mRNAs. It includes the masking of key sequences involved in the splicing of targeted exons by using antisense oligonucleotides (AON) that are complementary to exon definition sequences within a pre-mRNA. Provided herein are tc-DNA AONs that may be suitably employed for exon skipping through the masking of splice sites at intron/exon junctions, or mo

Problems solved by technology

The foremost consequence of DMD is that muscle fibers become particularly fragile and natural muscle activity provokes general damage in muscle tissue.
Mutations in the dystrophin gene result in a failure to produce dystrophin in striated muscles.
Others have point mutations or very small deletions or duplications that are difficult to identify.
Out-of-frame deletions or non-sense mutations that yield premature stop codons and subsequent abortion of translation result in dystrophin deficiencies characterized by severe phenotypes.
Several studies over the past 10 years support the benefit of steroid treatment (prednisone and deflazacort) in Duchenne boys, although a broad statistical evaluation has not yet been fully completed.
Recent developments have also provided evidence that stem cells from either bone marrow or vascular origins can target skeletal muscle through the systemic pathway, even though the extent of the genetic correction is still insufficient.
Although AAV vectors lack all viral genes, their cargo shipment is limited to 4.5 kb.
Although the proportion of revertant fibers increases with time, their number is unfortunately too low to confer a significant clinical benefit.
Nevertheless, the weakness of this approach is that it requires regular administration of the synthetic AOs, and systemic delivery has not been fully achieved.
Even so, producing “therapeutic” antisense RNA molecules in vivo poses many pr

Method used

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  • Tricyclo-dna antisense oligonucleotides, compositions, and methods for the treatment of disease
  • Tricyclo-dna antisense oligonucleotides, compositions, and methods for the treatment of disease
  • Tricyclo-dna antisense oligonucleotides, compositions, and methods for the treatment of disease

Examples

Experimental program
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Effect test

example 1

Use of Tricyclo-DNA Antisense Oligonucleotides to Rescue Dystrophin in Dystrophic Muscle Fibers

[0142]Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder that results from mutations in the gene encoding dystrophin. Out-of-frame deletions within the dystrophin gene that encode a truncated dystrophin protein deficiency lead to severe DMD phenotypes. Exon-skipping strategies using tricyclo-DNA (tc-DNA) antisense oligonucleotides (AON) were developed to permit the efficient rescue of out-of-frame dystrophin gene mutations thereby restoring the translational reading-frame and hence the production of functionally active dystrophin protein. Tc-DNA AON are described that, for example, hybridize to an exon 23 / intron 23 junction and interfere with pre-mRNA processing such that exon 23 is spliced out of the resulting processed mRNA. Alternatively, tc-DNA AON that hybridize to an exon 51 / intron 51 junction similarly interfere with pre-mRNA processing such that exon 51 is spliced...

example 2

The Mdx Mouse Model

[0143]The mdx mouse is a murine model of DMD that lacks the full length dystrophin protein, but retains all the smaller dystrophin isoforms. Bulfield et al., Proc. Natl. Acad. Sci. USA 81:1189-1192 (1984). The mdx mouse carries a non-sense mutation in exon 23 of the dystrophin gene, which precludes functional dystrophin synthesis (see, FIG. 3). Exon 23 partially encodes repeats R6 and R7 in which a C to T mutation creates a stop codon (TAA).

example 3

In Vitro Studies

[0144]This example demonstrates that mdx myotubes transfected with a 15-nucleotide tc-DNA AON designated M23D (+02−13) having the nucleotide sequence 5′-AACCTCGGCTTACCT-3′ undergo exon skipping at the downstream donor splice site of exon 23 such that dystrophin pre-mRNA is processed to mRNA that are deleted in exon 23.

[0145]The tc-DNA AON designated M23D (+02−13), was designed such that it hybridizes to the target sequence intron 22—ttttgag[GCTC . . . EXON 23 . . . TCAG]gtaagccgaggtttggcc—intron 23 at the exon 23 / intron 23 splice junction.

[0146]Mdx myotubes were transfected with tc-DNA AON M23D (+02−13) (1, 2 and 10 μg) with or without oligofectamine. One sample was left untreated, as a negative control. After 48 hours, cultures were harvested and mRNA was extracted using the RNeasy mini kit (Qiagen). mRNA was then reverse transcribed, as follows. Eight microliters of extracted RNA (500 ng to 1 μg) was mixed with 1 μL dNTP and 1 μL random hexamers, and the mixture w...

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Abstract

Provided are tricyclo-DNA (tc-DNA) AON and methods employing tc-DNA AON for modifying splicing events that occur during pre-mRNA processing. Tricyclo-DNA (tc-DNA) AON are described that may be used to facilitate exon skipping or to mask intronic silencer sequences and/or terminal stem-loop sequences during pre-mRNA processing and to target RNase-mediated destruction of processed mRNA. Tc-DNA AON described herein may be used in methods for the treatment of Duchenne Muscular Dystrophy by skipping a mutated exon 23 or exon 51 within a dystrophin gene to restore functionality of a dystrophin protein; in methods for the treatment of Spinal Muscular Atrophy by masking an intronic silencing sequence and/or a terminal stem-loop sequence within an SMN2 gene to yield modified functional SMN2 protein, including an amino acid sequence encoded by exon 7, which is capable of at least partially complementing a non-functional SMN1 protein; and in methods for the treatment of Steinert's Myotonic Dystrophy by targeting the destruction of a mutated DM1 mRNA comprising 3′-terminal CUG repeats.

Description

BACKGROUND OF THE DISCLOSURE [0001]1. Technical Field[0002]The present disclosure relates, generally, to synthetic antisense oligonucleotides (AON) and methods employing antisense oligonucleotides for modifying splicing events that occur during pre-mRNA processing or for down-regulating the expression of mutated mRNA that contain repeated sequences such as, for example, 3′ or 5′ CUG, CAG, and / or CCUG. More specifically, disclosed herein are tricyclo-DNA (tc-DNA) AON that are effective in facilitating exon skipping during pre-mRNA processing, in masking intronic silencer sequences and / or stem-loop sequences in pre-mRNA, and in targeting the RNase-mediated destruction of mRNA. Described herein are tc-DNA AON that may be used in methods for the treatment of Duchenne Muscular Dystrophy by skipping mutated exons, such as a mutated exon 23 or exon 51, within a dystrophin gene to restore functionality of a dystrophin protein. Also described are tc-DNA AON that may be used in methods for th...

Claims

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Application Information

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IPC IPC(8): A61K31/712A61P21/00A61P25/00C07H21/04C12N5/071
CPCA61K31/711A61K31/712C12N15/111C12N2320/33C12N2310/11C12N2310/3231C12N15/113A61P19/08A61P21/00A61P21/04A61P25/00A61P43/00
Inventor SCHUMPERLI, DANIELLEUMANN, CHRISTIANFURLING, DENISGARCIA, LUISVOIT
Owner INST NAT DE LA SANTE & DE LA RECHERCHE MEDICALE (INSERM)
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