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Methods for the production of functional protein from DNA having a nonsense mutation and the treatment of disorders associated therewith

a technology of functional proteins and nonsense mutations, which is applied in the field of production of functional proteins from dna having nonsense mutations and the treatment of disorders associated therewith, can solve the problems of many amino acid substitutions that do not have a gross effect on protein structure or function, major genome disruption, and inability to produce aberrant proteins in cells

Inactive Publication Date: 2016-07-14
PTC THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides nonsense codon suppressor agents that can be orally administered to a subject to prevent, treat, and manage a disease associated with a nonsense mutation in a gene. These agents have few adverse side effects and can be taken long-term without toxicities. The oral administration of these agents eliminates the need for a medical professional to administer them and improves the quality of life of the subject. The oral administration of these agents also allows for systemic therapy to all disease-affected organ sites. The invention provides a new treatment option for patients with nonsense mutations in genes.

Problems solved by technology

Mutations of the DNA sequence in which the number of bases is altered are categorized as insertion or deletion mutations (frameshift mutations) and can result in major disruptions of the genome.
These premature stop codons can produce aberrant proteins in cells as a result of premature translation termination.
The inserted amino acid will not necessarily be identical to the original amino acid of the wild-type protein; however, many amino acid substitutions do not have a gross effect on protein structure or function.

Method used

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  • Methods for the production of functional protein from DNA having a nonsense mutation and the treatment of disorders associated therewith
  • Methods for the production of functional protein from DNA having a nonsense mutation and the treatment of disorders associated therewith
  • Methods for the production of functional protein from DNA having a nonsense mutation and the treatment of disorders associated therewith

Examples

Experimental program
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Effect test

example 1

6.1 Example 1

Identification and Characterization of Compounds that Promote Nonsense Mutation Suppression and / or Modulate Translation Termination

[0434]Compounds that modulate premature translation termination and / or nonsense-mediated mRNA decay can be identified by a number of techniques. For example, methods for screening compounds that modulate the post-transcriptional expression of any gene with a premature translation stop codon are described in International Patent Publication No. WO 01 / 44516 A2, incorporated by reference herein in its entirety. In one example, a mRNA with a premature termination codon is translated in vitro and is used to screen a library of test compounds. In another example, the mRNA with a premature termination codon is a reporter gene with a premature termination codon.

[0435]Two assays were developed for use in high throughput screens to identify small molecules that promote nonsense mutation suppression. Each assay utilizes luciferase because it is a funct...

example 2

6.2 Example 2

Characterization of Compounds that Increase Nonsense Mutation Suppression and Produce Functional Protein

[0436]To determine in vivo activity, a stable cell line harboring the UGA nonsense-containing luciferase gene is treated with test compounds. Cells are grown in standard medium supplemented with 1% penicillin-streptomycin (P / S) and 10% fetal bovine serum (FBS) to 70% confluency and split 1:1 the day before treatment. On the following day, cells are trypsinized and 40,000 cells are added to each well of a 96-well tissue culture dish. Serial dilutions of each compound are prepared to generate a six-point dose response curve spanning 2 logs (30 μM to 0.3 μM). The final concentration of the DMSO solvent remains constant at 1% in each well. Cells treated with 1% DMSO serve as the background standard, and cells treated with gentamicin serve as a positive control.

example 3

6.3 Example 3

Nonsense Suppressors Alter the Accessibility of the Chemical Modifying Agents to Specific Nucleotides in the 28 S rRNA

[0437]Previous studies have demonstrated that gentamicin and other members of the aminoglycoside family that decrease the fidelity of translation bind to the A site of the 16S rRNA. By chemical footprinting, UV cross-linking and NMR, gentamicin has been shown to bind at the A site (comprised of nucleotides 1400-1410 and 1490-1500, E. coli numbering) of the rRNA at nucleotides 1406, 1407, 1494, and 1496 (Moazed & Noller, 1987, Nature 327(6121):389-394; Woodcock et al., 1991, EMBO J. 10(10):3099-3103; and Schroeder et al., 2000, EMBO J. 19:1-9.

[0438]Ribosomes prepared from HeLa cells are incubated with a test compound (at a concentration of 100 μM), followed by treatment with chemical modifying agents (dimethyl sulfate [DMS] and kethoxal [KE]). Following chemical modification, rRNA is phenol-chloroform extracted, ethanol precipitated, analyzed in primer ex...

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Abstract

The present invention relates to functional proteins encoded by nucleic acid sequences comprising a nonsense mutation. The present invention also relates to methods for the production of functional proteins encoded by nucleic acid sequences comprising a nonsense mutation and the use of such proteins for prevention, management and / or treatment of diseases associated with a nonsense mutation(s) in a gene.

Description

[0001]This application claims the benefit of U.S. provisional application Ser. No. 60 / 787,333, filed Mar. 30, 2006 and U.S. provisional application Ser. No. 60 / 813,085, filed Jun. 12, 2006, the disclosures of which are incorporated by reference herein in their entireties.1. FIELD OF INVENTION[0002]The present invention relates to functional proteins encoded by nucleic acid sequences comprising a nonsense mutation. The present invention also relates to methods for the production of functional proteins encoded by nucleic acid sequences comprising a nonsense mutation and the use of such proteins for prevention, management and / or treatment of diseases associated with a nonsense mutation(s) in a gene.2. BACKGROUND OF THE INVENTION[0003]Gene expression in cells depends upon the sequential processes of transcription and translation. Together, these processes produce a protein from the nucleotide sequence of its corresponding gene.[0004]Transcription involves the synthesis of RNA from DNA b...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/4245
CPCA61K31/4245A61K31/40A61K31/41A61K31/4162A61K31/44A61K31/46A61K31/00A61P1/00A61P11/00A61P11/02A61P13/12A61P17/16A61P21/00A61P21/04A61P25/00A61P25/16A61P25/28A61P27/02A61P3/04A61P3/06A61P43/00A61P5/00A61P5/10A61P5/14A61P7/00A61P7/04A61P9/10
Inventor ALMSTEAD, NEILCHEN, GUANGMINGHIRAWAT, SAMITHWANG, PETER SEONGWOOKARP, GARY M.KENNEDY, PAULMILLER, LANGDONMOON, YOUNG-CHOONREN, HONGYUTAKASUGI, JAMES J.WELCH, ELLEN M.WILDE, RICHARD G.
Owner PTC THERAPEUTICS INC
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