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Methods to reprogram splice site selection in pre-messenger rnas

a splice site and pre-messenger technology, applied in the field of splice site selection, can solve the problems of loss of tumor suppressor activity, resistance to chemotherapeutic drugs, and decreased apoptosis in tumors, and achieve the effect of altering the use of splice sites

Inactive Publication Date: 2006-03-16
UNIV DE SHERBROOKE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention is about a method of controlling splicing of pre-mRNA molecules in a cell or cell extract. It involves using a hybrid oligonucleotide-protein conjugate that contains a nucleic acid sequence that is complementary to a specific region upstream of the splice site in the pre-mRNA molecule. This conjugate can interfere with the splicing process and either increase or decrease the production of mRNA and protein products. The method can be used for therapeutic purposes and can also be used to study the function of specific genes. The invention provides a tool for research and development of new treatments for genetic diseases."

Problems solved by technology

For example, the inappropriate inclusion of exons in BIN1 mRNA results in the loss of tumor suppressor activity.
For example, overexpression of Bcl-xL is associated with decreased apoptosis in tumors, resistance to chemotherapeutic drugs, and poor clinical outcome.
Accordingly, reprogramming the alternative splicing of any of these proteins has the potential to affect the function of each of these proteins.
The presence of intronic sequences that resemble splicing signals may also promote a multitude of weaker and non-productive interactions that will decrease the pairing efficiency of correct splice sites.
These potential problems may explain why short introns are more prevalent in highly expressed genes.
Understanding how the removal of long introns occurs efficiently and accurately remains a tremendous challenge for which very little experimental work has been accomplished.

Method used

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  • Methods to reprogram splice site selection in pre-messenger rnas
  • Methods to reprogram splice site selection in pre-messenger rnas
  • Methods to reprogram splice site selection in pre-messenger rnas

Examples

Experimental program
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Effect test

example 1

Effects of Splice Site Interference Using Oligonucleotide Versus Protein Binding

[0144] To determine the relative ability of oligonucleotide binding versus directed protein binding to interfere with splice site selection, an in vitro splicing assay was developed in HeLa cell extracts. This assay utilized a model pre-mRNA substrate (hereafter referred to as “553 or C5′− / − pre-mRNA”) containing competing 5′ splice sites taken from hnRNP A1 exon 7 and exon 7B. The 553 pre-mRNA was radioactively labeled with 32p and incubated in a HeLa nuclear extract for two hours, and then total RNA was isolated and fractionated on acrylamide / urea gels. Oligonucleotides were resuspended in water and added to the splicing mixtures containing extracts and target pre-mRNA at indicated concentrations. Normally, the pre-mRNA was spliced predominantly to the internal (proximal) 5′ splice site of exon 7B. However, if the proximal 5′ splice site was somehow blocked, then the distal site from exon 7 was used. ...

example 2

Effect of Protein Binding at Different Positions

[0147] The effect of targeting the binding of a protein in the vicinity of a 5′ splice site with the goal of interfering with its use through steric hindrance was also tested. Although a few natural cases of this type of splicing control exist, it was intended to ascertain the parameters that are associated with such an effect using a pre-mRNA that contain two competing 5′ splice sites. Using the C5′− / − pre-mRNA derived form the hnRNP A1 gene, the effect of targeting the binding of the bacteriophage MS2 coat protein close to the proximal 5′ splice site was tested. A high-affinity MS2 binding site was inserted at various positions (−46, −37, −26, −17, +15, +23 and +31) upstream or downstream of the proximal 5′ splice junction (FIG. 2A) and the in vitro splicing of the resulting pre-mRNAs was carried out in a HeLa extract supplemented in the presence or the absence of the recombinant GST-MS2 protein. As seen in FIG. 2B, positioning GST-...

example 3

Effects of Targeted Protein Binding in Trans on Splice Site Interference in vitro

[0149] The applicants also determined that targeting protein binding to promote interference did not require that the binding site be present in cis (i.e., on the pre-mRNA itself. Indeed, the binding site was effective when provided in trans using an oligonucleotide that contains the protein binding site and a portion complementary to the target sequence. A series of antisense oligos complementary to a portion of the C5′− / − pre-mRNA −4 to −23 upstream of the proximal 5′ splice site (FIG. 4A) was designed. The C5-M4A1 oligo contains a16 nt-long non-hybridizing 5′ extension made of DNA and carrying one high-affinity binding site for the hnRNP A1 / A2 proteins (TAGGGA). The C5-M4A1W contains the winner RNA sequence for optimal hnRNP A1 binding. A mutated version of this oligo (C5-M4A1M) harboring two GGG to CGC mutation was used as a control. Oligos carrying a non-related 16 nt-long tail (C5-M4CT) or lackin...

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Abstract

The present invention relates to a method of modulating splice site selection, splicing and alternative, the method comprising the step of hybridizing an oligonucleotide-protein conjugate to a target pre-mRNA molecule in a cell or cell extract, wherein the oligonucleotide-protein conjugate comprises an oligonucleotide moiety which comprises at least two distinct sequence elements: (i) a nucleic acid sequence that is complementary to a specific region upstream of the splice site in the target pre-mRNA molecule; and (ii) an extension containing a protein binding site sequence element for covalently binding a protein; wherein the protein moiety comprises a protein capable of modulating splicing of the splice site upon binding with the protein binding site.

Description

BACKGROUND OF THE INVENTION [0001] (a) Field of the Invention [0002] This invention relates to splice site selection, a process required for the generation of mRNAs encoding different proteins. [0003] (b) Description of Prior Art [0004] The completion of genome sequencing efforts for the Drosophila, the mouse and the human genomes has led to the conclusion that complex organisms have a smaller than expected set of protein-coding genes. In contrast, the full complement of proteins found in complex animals is much more diverse. While post-translational modifications probably account for a good fraction of protein diversity, the principal mechanism used to generate protein diversification is likely due to alternative pre-mRNA splicing mechanisms which act post-transcriptionally (Maniatis, T and Tasic, B, (2002) Nature 418:236, Black, D. L., (2003), Annu. Rev. Biochem. 72:291-336). [0005] Recent estimates based on analyses of Expressed Sequences Tags (ESTs) corresponding to mRNAs predic...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C12Q1/68A61K38/00C12N15/09A61K38/16A61P7/04A61P7/06A61P21/02A61P21/04A61P25/28A61P27/02A61P31/18A61P35/00C07K14/08C07K14/47C12N15/113C12Q1/02
CPCA61K38/00A61K48/00C07K2319/00C12N15/113C12N2310/321C12N2310/3521A61P21/02A61P21/04A61P25/28A61P27/02A61P31/12A61P31/18A61P35/00A61P7/04A61P7/06
Inventor CHABOT, BENOITVILLEMAIRE, JONATHANELELA, SHERIFNASIM, FAIZ-UL
Owner UNIV DE SHERBROOKE
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