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Delivery of Oligonucleotide-Functionalized Nanoparticles

Inactive Publication Date: 2010-09-16
NORTHWESTERN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0032]FIG. 4 depicts the duplex invasion scheme. A) Schematic of invasion of a duplex (fluorescein and adjacent dabcyl at terminus of duplex) by nanoparticle thereby releasing fluorescence signal. B) Results demo

Problems solved by technology

In particular, siRNA treatment may target a single point mutation in a gene, while small molecule therapy to date does not precisely distinguish between mutant and normal gene products.
As with delivery of many proteins, degradation of nucleic acids and poor bioavailability from the gastrointestinal tract are major hurdles to the oral delivery of siRNAs.
Even with intravenous delivery, conventional siRNA is rapidly degraded by serum factors and does not reach its targets.
Mechanical approaches, such as ultrasound, laser and injection, have been used to facilitate penetration through the mouse stratum corneum and drive siRNA into skin, but require specialized equipment, limit the area of delivery, and potentially harm the skin.
Thus, traversing this layer to transfer sufficient amounts of oligonucleotides has been a challenge.

Method used

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  • Delivery of Oligonucleotide-Functionalized Nanoparticles
  • Delivery of Oligonucleotide-Functionalized Nanoparticles
  • Delivery of Oligonucleotide-Functionalized Nanoparticles

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Nanoparticles

[0184]Citrate-stabilized gold nanoparticles (from 1-250 nm) are prepared using published procedures [G. Frens, Nature Physical Science. 1973, 241, 20]. While a 13 and 5 nm size is used in this example, other examples include nanoparticles in size from 1 nm to 500 nm. Briefly, hydrogen tetrachloroaurate is reduced by treatment with citrate in refluxing water. The particle size and dispersity can be confirmed using transmission electron microscopy and uv / vis spectrophotometry. Thiolated oligonucleotides are synthesized using standard solid-phase phosphoramidite methodology [Pon, R. T. Solid-phase supports for oligonucleotide synthesis. Methods in Molecular Biology (Totowa, N.J., United States) (1993), 20 (Protocols for Oligonucleotides and Analogs), 465-496]. The thiol-modified oligonucleotides are next added to 13±1 and 5 nm gold colloids at a concentration of 3 nmol of oligonucleotide per 1 mL of 10 nM colloid and shaken overnight. After 12 hours, sodium ...

example 2

Oligonucleotide Modified Nanoparticle Conjugate Methods

[0185]Oligonucleotide design in this example includes two possible mechanisms of action. First, a sequence was designed using the published plasmid sequence that would preferentially hybridize to the sense strand of the promoter site for the Ampicillin resistance (AmpR) gene β-lactamase. This would sensitize the bacteria to ampicillin by taking advantage of the preferential hybridization of the conjugate (imparted by more favorable binding constant and / or intracellular concentration of the particles) to the promoter sequence of AmpR in the bacterial genome. This would prevent the promoter complex from binding to its target site and prevent transcription of the mRNA transcript (Amp resistance gene), therefore sensitizing the bacteria to ampicillin. The sequences used were 5′-AT TGT CTC ATG AGC GGA TAC ATA TTT GAA AAA AAA AAA A-SH-3′ (SEQ ID NO: 1) and 5′-AT TGT CTC ATG AGC GGA TAC AAA AAA AAA A-SH-3′ (SEQ ID NO: 2).

[0186]A second...

example 3

Oligonucleotide Modified Nanoparticle Conjugates Achieve Transcriptional Knockdown

[0190]An additional strategy was employed to examine transcriptional knockdown in a plasmid derived Luciferase gene. This model was used to demonstrate site-selective gene knock down by differentiating Luciferase knockdown from a separate region on the plasmid encoding Renilla expression. To assay this effect the Dual-Luciferase Reporter Assay System (Promega) was used. The strategy employed for this model was to block formation of a full mRNA transcript of the luciferase gene. This results in diminution of luciferase signal in relation to renilla. The sequence used for this was 5′-CCC GAG CAA CGC AAA CGC AAA AAA AAA AA-SH-3′ (SEQ ID NO: 4). Alternatively, one could use a strategy similar to that used above to block the promoter complex from binding its target site. In this example, 5 nm particles were used. The resulting knockdown after 12 hours was 59% using 300 nM concentration of particles (p value...

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Abstract

The present invention relates to compositions and methods for delivering an oligonucleotide-functionalized nanoparticle.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims the priority benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61 / 187,759, filed Jun. 17, 2009, and is a continuation-in-part of U.S. application Ser. No. 12 / 684,836, filed Jan. 8, 2010 which claims the priority benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61 / 143,293, filed Jan. 8, 2009, and U.S. Provisional Application No. 61 / 169,384, filed Apr. 15, 2009, the disclosures of all of which are incorporated herein by reference in their entirety.STATEMENT OF GOVERNMENT INTEREST[0002]This invention was made with government support under Grant Numbers 5DP1 OD000285 and U54 CA0119341, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.FIELD OF THE INVENTION[0003]The present invention is directed to oligonucleotide-modified nanoparticle (ON-NP) conjugates and methods of inhibiting bacterial protein production. The invention also re...

Claims

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Application Information

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IPC IPC(8): A61K9/14A61K48/00A61P35/00A61P29/00A61P31/00A61P17/00
CPCA61K9/0014A61K9/5094A61K47/48015A61K47/48861B82Y5/00A61K31/00A61K47/52A61K47/6923A61P17/00A61P29/00A61P31/00A61P35/00
Inventor MIRKIN, CHAD A.PALLER, AMY S.GILJOHANN, DAVID A.
Owner NORTHWESTERN UNIV
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