Antisense oligonucleotide directed removal of proteolytic cleavage sites, the hchwa-d mutation, and trinucleotide repeat expansions

Abstract

Described are methods for removing a proteolytic cleavage site, the HCHWA-D mutation or the amino acids encoded by a trinucleotide repeat expansion from a protein comprising providing a cell that expresses pre-mRNA encoding the protein with an anti-sense oligonucleotide that induces skipping of the exonic sequence that comprises the proteolytic cleavage site, HCHWA-D mutation or trinucleotide repeat expansion, respectively, the method further comprising allowing translation of mRNA produced from the pre-mRNA.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation-in-part of U.S. patent application Ser. No. 13 / 814,203, filed Apr. 12, 2013, pending, which application is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT / NL2011 / 050549, filed Aug. 4, 2011, designating the United States of America and published in English as International Patent Publication WO 2012 / 018257 A1 on Feb. 9, 2012, which claims the benefit under Article 8 of the Patent Cooperation Treaty and under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No. 61 / 370,855, filed Aug. 5, 2010, and to European Patent Application Serial No. 10172076.1, filed Aug. 5, 2010, the entire disclosure of each of which is hereby incorporated herein by this reference.STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e)—SEQUENCE LISTING SUBMITTED AS A TXT AND PDF FILES[0002]Pursuant to 37 C.F.R. §1.821(c) or (e), files containing a TXT version and a PDF version of the Sequen...

AI Technical Summary

Benefits of technology

[0016]Successful allele-specific reduction of the mutant ataxin-3 transcript was shown using lentiviral shRNAs directed against a single nucleotide polymorphism (SNP) in the ATXN3 gene in vitro and in vivo. However, this approach is limited to SCA3 patients carrying a heterozygous SNP in the ATXN3 gene. The disclosure provides a novel way to reduce toxicity of the ataxin-3 protein through protein modification. Using AONs, it is possible to mask sequences in the pre-mRNA from the splicing machinery resulting in exclusion of the targeted exon, either partially or in its entirety. If the reading frame remains intact, subsequent translation yields an internally truncated protein. This has the major advantage that a polyQ-containing part of the protein is removed, while maintaining global ataxin-3 protein levels. This strategy can be used to remove the trinucleotide repeats in other proteins as well.

[0038]While not wishing to be bound by theory, removal of the exon containing the HCHWA-D mutation is thought to reduce the formation or slow the progression of amyloid protein deposits in cerebral blood vessel walls.

[0040]The methods hereof are particularly useful for removing proteolytic cleavage sites, the HCHWA-D mutation, and the amino acids encoded by trinucleotide repeat expansions from proteins. It does not require removal or modification of the gene itself, but rather, prevents the incorporation of the genetic code for the proteolytic cleavage site, HCHWA-D mutation, or trinucleotide repeat expansions into the coding region of the protein in the mature mRNA. In this way, the process is reversible. The oligonucleotide has a finite life span in the cell and, therefore, has a finite effect on the removal. Another advantage is that the removal is not absolute. Not all pre-mRNA coding for the target protein that is generated by the cell is typically targeted. It is possible to achieve high levels of skipping. The skipping efficiency depends, for instance, on the particular target, the particular exon sequence to be skipped, the particular AON design, and / or the amount of AON used. Skipping percentages are typically expressed as the ratio of mRNA that does not have the coding part of the proteolytic cleavage site (skipped mRNA) versus the sum of skipped mRNA and unmodified mRNA coding for the unmodified target protein (unmodified mRNA). The possibility of tailoring the percentage of skipping is advantageous; for instance, when the unmodified protein is associated with a toxic phenotype but also has a positive function to perform that is not performed (as well) by the modified protein. By removing the proteolytic cleavage site, HCHWA-D mutation, or the amino acids encoded by trinucleotide repeat expansions only from a fraction of the protein formed, it is possible to reduce the toxic property, while leaving the positive or desired function of the unmodified protein at least partially intact.

[0044]In contrast to the previous uses for exon-skipping, provided is a method for removing a proteolytic cleavage site, HCHWA-D mutation, or the amino acids encoded by a trinucleotide repeat expansions in order to treat an individual, restore function to a protein, or reduce toxicity of a protein. The methods and oligonucleotides described herein are particularly useful for removing proteolytic cleavage sites, HCHWA-D mutation, or the amino acids encoded by trinucleotide repeat expansions from a protein, wherein the protein is involved in a neurodegenerative disorder.

Problems solved by technology

Although this can be done in the laboratory, it is difficult to implement such strategies in the clinic, if only because gene therapy applications that rely on the introduction of a complete gene are, at present, not very efficient, and the original gene associated with the problem is not removed.

Images