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Targeting cells with altered microrna expression

a technology of microrna and target cells, which is applied in the direction of nerve system cells, drug compositions, line-transmission, etc., can solve the problems of inability to restrict the expression of the delivered gene to the desired tissue or type of cell, and inability to control the expression of the therapeutic gene, so as to improve cell survival or rescue, improve cell proliferation, and modulate the development of cells

Inactive Publication Date: 2012-07-26
SOUTHERN ADELAIDE HEALTH SERVICE - FLINDERS MEDICAL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for modulating the development of a cell by introducing a nucleic acid with the capacity to bind to a specific microRNA in the cell. This method can be used for genetic engineering and therapeutic gene transfer. By targeting specific microRNAs in cancer cells or pre-cancerous cells, the method can lead to the ablation of the cell or the improvement of its characteristics for gene therapy purposes. The invention can also be used to target specific microRNAs in stem cells for differentiation or gene therapy purposes. The level of activity and concentration of the nucleic acid in the cell can be effectively targeted by introducing a nucleic acid with the capacity to bind to a specific microRNA. This method can be used for genetic engineering and therapeutic gene transfer.

Problems solved by technology

Targeted gene expression is one of the most difficult and important goals in the development of effective therapies for a variety of disorders, including cell proliferative disorders such as cancer.
However, one major limitation of current gene therapy protocols has been the inability to control expression of the therapeutic gene, and in particular, the inability to restrict expression of the delivered gene to the desired tissue or type of cell.

Method used

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  • Targeting cells with altered microrna expression
  • Targeting cells with altered microrna expression
  • Targeting cells with altered microrna expression

Examples

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example 1

RNA Isolation From Tissue Samples and Cell Lines

[0234]Cell lines may be maintained in an appropriate medium. Colorectal tumors and the corresponding normal mucosae may be obtained from fresh surgical resections, following informed consent from patients, and then classified according to standard histopathological classification methods.

[0235]RNA may isolated from cell lines, using Trizol reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. RNA may be purified from colorectal tissues using the procedure of Chomczynski, P. and Sacchi, N. (1987). Anal. Biochem. 162: 156-159.

example 2

Cloning of MicroRNAs miRNAs may be cloned essentially as described by Elbashir et al. (2001) Genes Dev. 15: 188-200, except that nucleic acids may be electroeluted from acrylamide gel slices using the Biotrap system (Schleicher and Schuell GmbH, Dassel, Germany). Briefly, small RNA fractions of between 18 and 26 bases may be size selected on a denaturing polyacrylamide gel. Adapter oligonucleotides, containing EcoRI restriction sites, may then be directionally ligated to the RNA molecules. The adapter-ligated RNA may then be amplified by RT-PCR. Concatamerized fragments, containing multimers of religated, EcoRI-digested PCR products, between 200 and 650 bp, are size selected on an agarose gel and recovered by electroelution. The concatamers may then be end-repaired and dA-tailed with Taq DNA polymerase, then cloned into pGEM T-easy (Promega, Madison, Wis.) or pTOPO (Invitrogen) according to the manufacturers' instructions. Plasmid inserts from the resultant colonies may be analyzed ...

example 3

Northern Analysis

[0236]Total RNA (20 μg) may be separated on a 15% denaturing polyacrylamide gel. Loadings are visualized by ethidium bromide staining. The RNA may then be transferred to Hybond N+ nylon membrane by semi-dry blotting (OWL Separation Systems, Portsmouth, N.H.). Probes may be generated by T4 Polynucleotide Kinase (New England Biolabs, Beverly, Mass.) mediated end-labeling of DNA oligonucleotides with [γ-32P]ATP. To increase the specific activity of the probes, the miRNA sequence may be concatamerized as a trimer of direct repeats, then cloned into pGEM T-easy and the insert amplified using PCR with M13 forward and reverse primers. Antisense probes may then be synthesized using Taq polymerase-generated linear amplification from the Sephadex G-50-purified PCR products to incorporate multiple [γ-32P]dCTP bases.

[0237]Filter hybridization may be performed in QuikHyb Solution (Stratagene, La Jolla, Calif.) containing 106 cpm / ml probe for 1 h, with washes, as per the manufact...

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Abstract

The present invention relates to a method of modulating development of a cell. The method includes the step of introducing into the cell a nucleic acid with the capacity to modulate development of the cell, the nucleic acid including a target site for binding of a microRNA, wherein the activity and / or concentration of the microRNA in the cell results in a level of activity and / or concentration of the nucleic acid in the cell sufficient to modulate development of the cell.

Description

RELATED APPLICATIONS[0001]This application claims priority to International Patent Application No. PCT / AU2006 / 000750, International Filing Date Jun. 2, 2006, entitled Targeting Cells with Altered MicroRNA Expression; and claims priority to U.S. Provisional Application Ser. No. 60 / 687,547 filed Jun. 3, 2005, both of which are hereby incorporated by reference.FIELD OF THE INVENTION[0002]The present invention relates to a method of modulating the development of a cell with altered microRNA activity, and to a method of preventing and / or treating a disease, condition or state associated with altered microRNA activity.[0003]The present invention also relates to nucleic acids having a binding site for a microRNA, cells and animals including such nucleic acids, and compositions including the nucleic acids.BACKGROUND OF THE INVENTION[0004]Targeted gene expression is one of the most difficult and important goals in the development of effective therapies for a variety of disorders, including c...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/7088C12N5/09C12N5/0735C12N5/0793C12N5/076C12N15/11C12N15/33C12N15/38C12N15/54C12N15/53C12N15/57C12N15/19C12N15/63A61P35/00A61P35/02A61P31/18A61P31/14A61P31/22A61P29/00A61P1/00C12N5/071
CPCH04B3/548H04B2203/547H04B3/56A61P1/00A61P29/00A61P31/14A61P31/18A61P31/22A61P35/00A61P35/02
Inventor MICHAEL, MICHAEL ZENON
Owner SOUTHERN ADELAIDE HEALTH SERVICE - FLINDERS MEDICAL CENT
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