A modified human U1snRNA molecule, a gene encoding for the modified human U1snRNA molecule, an expression vector including the gene, and the use thereof in gene therapy

An expression vector, human technology, applied in the field of modified human snRNA molecules to achieve highly selective effects

Inactive Publication Date: 2013-09-18
UNIV FERRARA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the relative conservation of the 5' splice site and the attendant risk of interfering with the maturation of transcripts generated from otherwise functional wild-type genes, this approach presents a somewhat nonspecific effect of therapeutic snRNA molecules on the target gene.

Method used

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  • A modified human U1snRNA molecule, a gene encoding for the modified human U1snRNA molecule, an expression vector including the gene, and the use thereof in gene therapy
  • A modified human U1snRNA molecule, a gene encoding for the modified human U1snRNA molecule, an expression vector including the gene, and the use thereof in gene therapy
  • A modified human U1snRNA molecule, a gene encoding for the modified human U1snRNA molecule, an expression vector including the gene, and the use thereof in gene therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Generation of modified U1snRNA

[0037] The modified U1snRNA was generated by the following procedure: the plasmid containing the wild-type U1-snRNA gene, that is, the unmodified U1-snRNA, was digested with BglII and BclI restriction enzymes. The sequence formed between the two restriction sites is replaced with a double-stranded oligonucleotide containing the binding sequence. The following Table 1 describes the forward sequence and reverse sequence of each oligonucleotide, and the resulting modified U1-snRNA is named after the oligonucleotide used.

[0038] and, figure 2 A schematic diagram of U1snRNA gene elements is shown. The cloning strategy for preparing different modified U1snRNA is indicated. figure 2 It shows the U1snRNA gene inserted into the plasmid vector (pGEM) with the promoter elements DSE and PSE, the U1snRNA coding region (in the middle), and the 3'processing cassette. The transcription start site is indicated by an arrow. The sequence betw...

Embodiment 2

[0043] Example 2: Transfection of microgenes into cultured cells and analysis of splicing products

[0044] The inclusion vector is inserted into the cell by transient transfection with Lipofectamine (Liposome). After extracting total cellular RNA with Trizol, RNA was analyzed by RT-PCR with specific primers.

[0045] The reaction takes place in two steps: reverse transcription of RNA into a cDNA strand by reverse transcriptase using random primers as a template, and amplification of the resulting cDNA by DNA polymerase.

[0046] The PCR reaction was performed in a final volume of 25μl of a mixture containing:

[0047] -5μl AMV / Tfl5x buffer, suitable for the above two enzymes to function correctly;

[0048] -1μl of 10mM dNTP mixture;

[0049] -50pmol forward primer and 50pmol reverse primer;

[0050] -2μl of 25mM MgSO 4 ;

[0051] -2μl of RNA extracted from cells;

[0052] -1μl AMV-RT (0.1μ / μl), 1μl Tfl DNA polymerase;

[0053] -Appropriate amount of ultrapure H 2 O

[0054] The reverse tra...

Embodiment 3

[0056] Example 3: Exons near the donor site of coagulation factor IX associated with hemophilia B Mutations and mutations in the polypyrimidine sequence upstream of the exon 5 receptor site

[0057] In the factor IX gene (F9), the mutation of exon at position -2 in the donor site, and the mutation at positions -8 and -9 in the acceptor site of exon 5 are the same as the type B Hemophilia is associated. It is interesting to note that the mutation at position-2 in the exon is a synonymous mutation and does not change the coding sequence, but causes the exon to be missed and therefore it is classified as a splicing mutation. Mutations at positions -8 and -9 in the receptor site also caused the omission of exon 5.

[0058] Table 2 shows the mutations in question, which were identified in patients with hemophilia B (Haemophilia B International Database). Nucleotides belonging to exon 5 are shown in capital letters, while those belonging to introns are shown in lower case. Each posi...

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Abstract

A modified human U1snRNA molecule is described, the target sequence of which is located in a region of the pre-mRNA of the target gene comprised between 2 and 50 base pairs downstream of an exon / intron junction site, which is capable of restoring the correct splicing of a target gene of therapeutic interest bearing a mutation which induces exon skipping and resulting in a genetic disease. Modified human U1snRNA molecules are described by way of example for the correction of diseases associated with exon skipping, such as spinal muscular atrophy, hemophilia B, and cystic fibrosis.

Description

Technical field [0001] The present invention relates to a modified human snRNA molecule (hereinafter designated as Exon-specific U1-ExSpeU1), which is suitable for use in gene therapy methods. Specifically, the present invention relates to snRNA molecules capable of correcting abnormal splicing processes caused by gene mutations and associated with human diseases that are usually very serious with different medical histories. Background technique [0002] Many human genetic diseases (about 15%) are caused by genetic mutations, which cause diseases by interfering with the intracellular maturation of the correct messenger RNA, impairing subsequent accurate protein biosynthesis and inducing the synthesis of non-functional proteins. Generally, point mutations that cause splicing defects involve recognizing gene sequences that are critical to the primary transcript for the machinery responsible for processing the primary transcript. Donor and acceptor sites located at the exon-intron...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11A61K48/00A61P43/00A61P7/04A61P21/00C12N15/63
CPCA61P3/00A61P7/04A61P21/00A61P43/00C12N15/111C12N15/113C12N15/1137C12N15/1138C12N2310/11C12N2320/33C12N2330/51C12Y304/21022C12Y306/03049
Inventor 佛朗哥·帕加尼米尔科·皮诺蒂
Owner UNIV FERRARA
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