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Methods and compositions for manipulating translation of protein isoforms from alternative initiation start sites

a protein isoform and initiation start technology, applied in the field of antisense oligonucleotides and compositions containing antisense oligonucleotides, can solve the problems of reducing the translation efficiency of p53, affecting the expression quality and quantitative differences, and directing much effort to reconstitute or reactivate p53 expression, etc., to achieve the effect of enhancing the sensitivity of tumor cells to radiation therapy, enhancing the translation of a second

Inactive Publication Date: 2019-08-08
SAREPTA THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent provides methods and compositions for using antisense oligonucleotides to modulate p53 expression and increase sensitivity of tumor cells to chemotherapy and radiation therapy. The antisense oligonucleotides inhibit cell cycle progression and enhance sensitivity of tumor cells to chemotherapy and radiation therapy by reducing expression of full-length p53 and its mutant forms. The methods involve contacting the cells with an antisense oligonucleotide and a chemotherapy or radiation therapy, or a combination of both, to increase sensitivity of the cells and treat the cancer. The invention also provides specific antisense oligonucleotide sequences and compositions for reducing expression of full-length p53 and its mutant forms.

Problems solved by technology

In the case of alternate translation initiation start sites, a potential family of related proteins can be produced from a single transcript which can result in qualitative and quantitative differences in the expressed proteins.
As a result, much effort is directed at reconstituting or reactivating p53 expression in tumour cells that have undergone loss or missense mutation in p53.

Method used

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  • Methods and compositions for manipulating translation of protein isoforms from alternative initiation start sites
  • Methods and compositions for manipulating translation of protein isoforms from alternative initiation start sites
  • Methods and compositions for manipulating translation of protein isoforms from alternative initiation start sites

Examples

Experimental program
Comparison scheme
Effect test

example 1

pMOS Block P53 Expression and Generate P53 Isoforms

[0388]To investigate the effectiveness of PMOs in blocking p53 expression, wild-type p53 expressing breast cancer MCF7 cells were treated with PMOs and peptide-conjugated PMOs targeting the translation initiation site of p53 mRNA (M1), the splice acceptor site of exon 10 (E10SA) and the coding region within exon 10 (E10) (see Table A below for PMOs used in all experiments described herein).

TABLE Ap53 Targeting SequencesSEQPMOp53 RegionTargeting Sequence 5′-3′ID NO:M1AUG startGCGGCTCCTCCATGGCAGTGAC 3codonM40Met 40 AUGCATCAAATCATCCATTGCTTGG 4codonE2SDExon 2AGTTTCCATAGGTCTGAAAA 5E4SAIntron 3 / GIIIACTGTAGATGIGTGAA 6Exon 4E6SAExon 6CGGATAAGATGCTGAGGAGG 7E7SAExon 7GTTGTAGTGGATGGTGGTA 8E7SDExon 7CTGGAGTCTTCCAGTGTGAT 9E9SDExon 9AAGIGTGAAATATTCTCCATC10E10SAIntron 9 / GCGCTCACGCCCACGGATC11Exon 10E10Exon 10CCCTGCTCCCCCCTGGCTCC12(Internal)ControlScrambledTGCCATCAACATATCTTGATCG13ControlSD, slice donorSA, splice acceptorI, inosine residue

[0389]The p...

example 2

pMOS Block P53 Expression and P21 Induction after DNA Damage

[0394]To investigate the expression and function of p44 (ΔN-p53) and p48 (p53β / γ) isoforms in a broader range of cells under different stress conditions that activate p53, human H460 lung cancer cells were treated with doxorubicin, human HCT116 colon cancer cells were treated with 5-FU, and human OCI / AML-3 and OCI / AML-4 leukemia cells were treated with γ-radiation. All of these human cancer cell lines express wild-type p53 and respond to DNA damage by increasing p53 protein levels and activating expression of the p53-target gene, p21 (see FIG. 2).

[0395]As shown in FIG. 2, pre-treatment for 2 hours with peptide-conjugated M1 or E10SA prior to DNA damage led to the production of ΔN-p53 and p53β / γ, respectively, as seen previously in MCF7 cells. The levels of these stable p53 isoforms did not increase further after DNA damage. Both M1 and E10SA interfered with the accumulation of full-length p53 and with the induction of p21 a...

example 3

Evaluation of pMOS that Target P53 mRNA Splicing

[0397]In the following experiments, a panel peptide-conjugated PMOs was tested that target splice sites in exons 2, 4, 6, 7 and 9 of the human p53 gene (see Table A, in Example 1 above). With the exception of E2SD that targets the splice donor site of exon 2, the remaining PMOs exhibited variable effectiveness in suppressing p53 protein expression in H460 cells, revealed no new isoforms, and did not block expression of p21 in response to doxorubicin (see FIG. 3).

[0398]As shown in FIG. 3, E2SD produced a 44 kDa isoform that resembled ΔN-p53 and prevented the induction of p21 in response to doxorubicin treatment. This alternatively spliced transcript contains an intron 2-derived in-frame termination codon that arrests translation prematurely and was predicted to produce ΔN-p53 from codon 40. These findings suggest that E2SD and M1 produce identical truncated ΔN-p53 proteins through two different mechanisms: alternative splicing of exon 2...

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PUM

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Abstract

Provided herein are antisense oligonucleotides, compositions comprising antisense oligonucleotides, and methods for the use of antisense oligonucleotides in manipulating translation. Expression of isoforms of proteins expressed from different start codons of the same transcript are inhibited by antisense oligonucleotides, which may also enhance expression of non-target isoforms.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]The present application is a continuation application of U.S. application Ser. No. 14 / 232,858 filed on Apr. 17, 2014, which is a 371 application of PCT / US2012 / 046783 filed on Jul. 13, 2012, which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 61 / 508,558 filed on Jul. 15, 2011; each of which application is incorporated herein by reference in its entirety.STATEMENT REGARDING SEQUENCE LISTING[0002]The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 120178_493C1_SEQUENCE LISTING.txt. The text file is about 10.3 KB, was created on Apr. 12, 2019, and is being submitted electronically via EFS web.BACKGROUNDTechnical Field[0003]Provided herein are antisense oligonucleotides, compositions comprising antisense oligonucleotides, and me...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/113C12N15/11
CPCC12N15/113C12N15/111C12N2320/12C12N2310/3233C12N2310/11C12N2320/34C12N2310/3513
Inventor IVERSEN, PATRICK L.
Owner SAREPTA THERAPEUTICS INC
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