Modified oligomeric compounds comprising tricyclo-DNA nucleosides and uses thereof

A technology of oligomeric compounds and nucleosides, applied in the field of modified oligomeric compounds containing tricyclic DNA nucleosides and their uses, can solve the problems of acute toxicity, preferably long-term toxicity, etc.

Pending Publication Date: 2020-03-31
SYNTHENA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, replacement of oxygen in the phosphate backbone by sulfur, while significantly improving biodistribution, has also been shown to be associated with nonspecific protein binding and activation of the innate immune system (complement activation in particular), which can lead to acute Toxicity, at best long-term toxicity (Dirin and Winkler, Expert Opin. Biol. Ther. 2013)

Method used

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  • Modified oligomeric compounds comprising tricyclo-DNA nucleosides and uses thereof
  • Modified oligomeric compounds comprising tricyclo-DNA nucleosides and uses thereof
  • Modified oligomeric compounds comprising tricyclo-DNA nucleosides and uses thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0741] Compositions of the invention for treating Duchenne muscular dystrophy

[0742] Effectiveness evaluation

[0743] Adult mdx mice were injected weekly for 4 weeks with different 13-mer AONs targeting the donor splice site (M23D: +2-11) of exon 23 of dystrophin pre-mRNA (i.e. SY-0308, SY-0210 and SY-0299, SY-0343, SY-0442 and SY-0455 of the present invention) treatment. SY-0308 (also referred to herein interchangeably as "tcDNA-PO M23D") corresponds to p-CCTCGGCTTACCT-OH of SEQ ID NO: 1, wherein all nucleotides are tc-DNA, and all internucleoside linkages The group is a phosphodiester linking group, and p is the phosphate moiety at the 5' end. SY-0210 (also referred to herein interchangeably as "tcDNA-PS M23D") corresponds to p-CCTCGGCTTACCT-OH of SEQ ID NO: 1, wherein all nucleotides are tc-DNA, and all internucleoside linkages The group is a phosphorothioate linking group, and p is the phosphate moiety at the 5' end. Composition SY-0343 of the present invention, int...

Embodiment 2

[0765] Co-precipitation experiments and proteomics

[0766] Target oligonucleotides were synthesized as biotinylated conjugates for subsequent co-precipitation of potentially interacting serum proteins by using streptavidin beads. The biotinylated derivatives used in this study were SY-0440, SY-0427, SY-0446, SY-0448, SY-0445, SY-0443 and SY-0451, which are defined and characterized in Table 3 and as Figure 6 shows.

[0767] Biotinylated oligonucleotides immobilized on streptavidin beads were incubated in the presence of serum for 1 hour. Beads were collected by low speed centrifugation, washed and then dissolved in appropriate buffer for further protein analysis by SDS-PAGE, Orbitrap LC-MS / MS and SDS-PAGE / MALDI-TOF.

[0768] Figure 7SDS-PAGE analysis of proteins recovered from mouse and human sera is illustrated for the oligonucleotides used in this study. It appears that SY-0440 (i.e., tcDNA M23D with full PS) retains much more serum protein than its tcDNA-PO equivalent...

Embodiment 3

[0778] Stability of the composition of the present invention in human serum

[0779] The stability of preferred compositions of the invention in human serum was studied based on oligonucleotides SY-0343, SY-0442, SY-0299, SY-0450, SY-0455 and SY-0357. Table 3 defines and characterizes the compositions of the invention.

[0780] Prepare a mixture (total volume 80 µL) consisting of 40 µL of human serum, oligonucleotide stock solution, 20 µL of PBS (2x), and milliQ water in a 0.2 mL PCR vial such that the final concentration of oligonucleotide is 2 µM , and incubated at 37°C. Aliquots were taken after 4, 24 and 120 hours. Digest the mixture with proteinase K in a PCR vial for 2 h at 55 °C (80 µL of the mixture, 100 µL of proteinase K buffer 2x (200 mM Tris-HCl (pH 8.5), 400 mM NaCl, 10 mM EDTA, 0.4% SDS ), 20 µL proteinase K (20 mg / mL in 100 mM Tris-HCl (pH 8.5), 10 mM CaCl 2 middle). The mixture was then cooled to room temperature, centrifuged (13400 rpm, 10 minutes) and th...

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Abstract

The present invention relates to a composition comprising an oligomeric compound comprising one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides and one or more lipid moiety, wherein said one or more lipid moiety is covalently linked to said oligomeric compound either directly or via a spacer, and wherein preferably said oligomeric compound comprises from 5 to 40 monomer subunits, as well as pharmaceutical compositions thereof and their uses in the prevention or treatment of neuromuscular or musculoskeletal diseases such as Duchenne muscular dystrophy or Steinert disease.

Description

[0001] Description of the relevant application [0002] This application claims priority from European Application No. 17167427.8, filed April 20, 2017, and US Provisional Application No. 62 / 562124, filed September 22, 2017, both of which are incorporated herein by reference in their entirety. [0003] Background of the invention [0004] Antisense technology is an effective means for reducing the expression of specific gene products and thus finds use in therapeutic, diagnostic and research applications. In general, the rationale behind antisense technology is that an antisense compound (sequence of nucleotides or analogs thereof) hybridizes to a target nucleic acid and modulates gene expression activity or function (such as transcription and / or translation). Regardless of the specific mechanism, their sequence specificity makes antisense compounds attractive as tools for target validation and gene functionalization, as well as therapeutic agents that selectively modulate gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/02C07H21/04A61K31/712A61K31/7125A61P25/00
CPCC07H21/02C07H21/04A61K31/7125A61P25/00A61K31/712A61K31/711A61P21/00A61K47/544A61K31/7028C12N15/113
Inventor W.A.伦纳B.杜戈维奇R.贝尔托利尼
Owner SYNTHENA
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