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Gene Silencing by Single-Stranded Polynucleotides

a single-stranded polynucleotide, gene silencing technology, applied in the direction of instruments, biochemistry apparatus and processes, material analysis, etc., can solve the problems of unexpected potency, unsatisfactory anti-sense effects, and unsatisfactory anti-sense effects, so as to enhance treatment regimens

Inactive Publication Date: 2011-11-03
GENESEGUES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0009]Disclosed are methods for inhibiting expression of a targeted gene, as well as methods for activating both antisense and double-stranded RNase activity. The methods include the steps of providing a bifunctional single stranded chimeric polynucleotide comprising a 3′ RNA portion and a 5′ DNA portion, where the bifunctional single stranded chimeric polynucleotide is capable of specifically hybridizing to the RNA sequence of the target gene, and delivering the bifunctio...

Problems solved by technology

Thus a given disease state may not be a candidate for antisense therapy because the target tissue has insufficient RNase H activity.
The composition used in these studies, “Formula G” was optimized for uniform particle size distribution and unexpectedly potent.

Method used

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  • Gene Silencing by Single-Stranded Polynucleotides
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Examples

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example 1

Formulation of Single-Stranded, Bifunctional Nucleic Acid Molecules for In Vitro and In Vivo Nuclear Delivery to Target Cells

[0106]This example describes how illustrative colloidal formulations of diverse cargos may be generated to practice the present inventions. Nanocapsules for uptake / binding, expression and imaging studies were prepared by the “dispersion atomization” method described in U.S. Pat. No. 6,632,671, which is incorporated herein by reference in its entirety, with modifications as described herein. These particles are used for in vitro transfection studies in full serum-containing media. The cell penetrating peptide MPG for nuclear delivery is a 27-mer peptide, composed of the N-terminal domain of the HIV gp41 fusion sequence fused to the C-termain domain derived from the nuclear localization signal derived from the SV40 large-T antigen and is more fully described in U.S. Pat. No. 6,841,535 and used here according to Simeoni et al., (Nucleic Acids Research, 2003, Vol....

example 2

Nuclear Delivery Creates Significant Utility for Single-Stranded Nucleic Acid Drug Species

[0121]Cultures were prepared for confocal microscopy by precoating flame-sterilized glass coverslips, with a model tumor matrix (2:1 Tenascin:Fibronectin, Millipore CC065: Sigma F0895) at a concentration of 0.5 ug per sq. cm. in six-well plates overnight at room temperature. Cells were plated at 100,000 per well and treated 2-3 days later. Cultures were treated with 200 nM Fitc-labeled ds siRNA or oligos formulated as s50 or MPG protein nanoparticles at 50 nM as described in Example 1, Formula C-E or lipid complexes using DOTAP (Roche) per the manufacturer's instructions. Protein nanoparticles formulation utilizing nonendosomal uptake mechanisms are used to illustrate impact of nuclear delivery relative to conventional cytosolic delivery via endosomal escape following uptake by lipid complexes into clathrin-coated pits. Following treatment with either nanoparticles or lipid complexes, cultures ...

example 3

Incorporation of Functional Guide Strand Sequences into Nuclear-Delivered Bifunctional ss Chimeric Polynucleotides Provides Method for Executing Potent Protein Inhibition

[0130]Having observed that unmodified ss chimeric polynucleotides combined with nuclear delivery to target cells creates a bifunctional RNAseH and RNAi molecule (interacts with type III dsRNAses), we sought to identify a useful procedure for application of this potentially more potent strategy to the problem of variable gene silencing and protein inhibition. A bifunctional nucleic acid drug offers advantages over one with single functionality in being better able to maintain potency across tissue variations. We were particularly interested in identifying guidelines for sequence selection and medicinal chemistry constraints. Using the experimental microscopy paradigm described in Example 2 in combination with different timepoints and the three cell lines, with formulation as described in Example 1, Formulas C-E, a se...

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Abstract

The present invention relates to compositions and methods for concurrently activating antisense and double-stranded RNase (dsRNase) mechanisms for inhibiting expression of a targeted gene, by delivering a single stranded bifunctional chimeric DNA / RNA oligonucleotide optimized for siRNA activity as well as antisense activity, into the nucleus of a target cell.

Description

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0001]This invention was made in part with government support from the National Institutes of Health under Grant No. NIH GRANT R43CA119556. The United States Government may have certain rights in the invention.TECHNICAL FIELD[0002]The present invention relates to the field of gene silencing and concurrent activation of RNAi and antisense pathways in a cell, via a single-stranded polynucleotide delivered to the nucleus of the cell.BACKGROUND[0003]Oligonucleotide compounds have important therapeutic applications in medicine. Oligonucleotides can be used to silence genes that are responsible for a particular disease. Gene silencing prevents formation of a protein by inhibiting translation. Importantly, gene-silencing agents are a promising alternative to traditional small, organic compounds that inhibit the function of the protein linked to the disease. Antisense, small interfering RNAs (siRNAs), microRNAs (miRNAs), small h...

Claims

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Application Information

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IPC IPC(8): A61K31/7088C12N15/00C12Q1/68
CPCC12N15/111C12N15/113C12N2310/11C12N2310/14A61K31/7125C12N2310/321C12N2310/51C12N2310/315C12N2310/3521C12N2320/32G01N33/5308
Inventor UNGER, GRETCHEN M.
Owner GENESEGUES
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