Alhasan A.H.,Northwestern University |
Patel P.C.,AuraSense Therapeutics LLC |
Choi C.H.J.,Northwestern University |
Mirkin C.A.,Northwestern University
Small | Year: 2014
Exosomes are a class of naturally occurring nanomaterials that play crucial roles in the protection and transport of endogenous macromolecules, such as microRNA and mRNA, over long distances. Intense effort is underway to exploit the use of exosomes to deliver synthetic therapeutics. Herein, transmission electron microscopy is used to show that when spherical nucleic acid (SNA) constructs are endocytosed into PC-3 prostate cancer cells, a small fraction of them (<1%) can be naturally sorted into exosomes. The exosome-encased SNAs are secreted into the extracellular environment from which they can be isolated and selectively re-introduced into the cell type from which they were derived. In the context of anti-miR21 experiments, the exosome-encased SNAs knockdown miR-21 target by approximately 50%. Similar knockdown of miR-21 by free SNAs requires a ≈3000-fold higher concentration. Spherical nucleic acid (SNA) constructs can be naturally sorted into exosomes when endocytosed into PC-3 prostate cancer cells. The exosome-encased SNAs are secreted into the extracellular environment from which they can be isolated and selectively re-introduced into the cell type from which they were derived as potent microRNA regulation agents. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Agency: Department of Defense | Branch: Air Force | Program: SBIR | Phase: Phase I | Award Amount: 100.00K | Year: 2011
ABSTRACT: Polyvalent nanoparticle constructs have unique properties that make them ideal for gene regulation applications, while overcoming many of the challenges which have prevented oligonucleotides from being developed into viable therapies. Specifically, AuraSense is developing constructs which are highly resistant to nuclease digestion, have high binding constants for intracellular targets, and, uniquely, have exhibited high entry into every cell type tested to date (over 50 cell types including primary cells, tissues and neurons). The AuraSense constructs thus represent a significant advance in gene pathway regulation technology while displaying the characteristics of an ideal gene therapy system. Building on our initial success, we are proposing to develop optimal constructs for application in the control of gene and protein expression. Objective 1 experiments will be designed to demonstrate and optimize: 1) Ability to deliver conjugate nanostructures into both prokaryotic and eukaryotic cells, 2) Ability to deliver oligonucleotides including siRNA, DNA and modified nucleic acid structures, and 3) Qualitative and quantitative assessment of cellular entry. Objective 2 will determine the 1) Functional gene regulatory effect in prokaryotic and eukaryotic cells, 2) Biological compatibility and toxicity, and 3) Comparison with commercially available lipid and polymer systems (e.g. Lipofectin and Cytofectin). BENEFIT: AuraSense"s nanoparticle constructs have great potential as broadly effective, non-toxic agents for cellular transfection of genetic material. AuraSense nanoparticle technology has been developed in numerous proof-of-concept applications and has been the subject of consistent commercial interest to date. This platform stands to significantly advance transfection as a tool for life science researchers and for increasing the safety of troops in the field. AuraSense"s nanoparticle platforms are highly relevant to two markets: Chemical Transfection (as used for research applications) and Biodefense.
Agency: Department of Defense | Branch: Air Force | Program: SBIR | Phase: Phase II | Award Amount: 728.58K | Year: 2012
ABSTRACT: AuraSense is developing and commercializing NanoFlare technology as an enabling tool for detecting RNA levels in cells. Current methods for sensing of viral exposure require great amounts of virus particles to be present in blood, or extensive processing of blood to isolate virus-specific genomic markers. We will develop NanoFlare sensors that can penetrate through skin, enter the bloodstream and detect viral targets. Once inside blood cells, the sensors will signal the presence of virus-specific genomic markers. This signal can be detected by fluorescence spectroscopy. The flexibility of the NanoFlare platform will facilitate the tailoring of the NanoFlare to meet current and emerging threats. BENEFIT: AuraSense will demonstrate an approach for detection of viral RNA in living cells. This will be accomplished using non-invasive systemic delivery through the skin. NanoFlare technology could thus be adapted for field deployment, for example by creating a patch to systemically deliver NanoFlares. We envision that a NanoFlare patch could be used by the warfighter to determine viral infection by taking a finger prick blood sample, with a rapid readout using a simple fluorescence detector such as a UV light source. In the commercial space, such a point-of-care fluorescence detection can be used by doctors in the clinic for rapid PCR-free diagnostic assays.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 200.00K | Year: 2010
DESCRIPTION (provided by applicant): Under the auspices of this small business innovation research (SBIR) grant, AuraSense LLC will develop a novel nanotechnology, Nano-flares , for the detection of low abundance microRNA (miR) species in living cells in real-time. MicroRNAs are short (~22 nucleotides in length) non-protein coding RNA sequences that play a key role in regulating diverse cellular processes that occur, for instance, during development or during malignant transformation. Unique miRs are being evaluated as novel biomarkers for myriad diseases, and explored as novel therapeutic targets. No existing commercial technology is capable of detecting intracellular RNA target sequences, including miRs, in real-time in living cells. Nano-flares technology provides new opportunities for the detection of intracellular miR targets and will identify how these unique RNAs can be more effectively detected, improving our knowledge of miR function, and, ultimately targeted to improve human health. Nano-flare technology is based upon the unique conjugate properties of DNA oligonucleotides which can be densely loaded on the surface of gold nanoparticles (DNA-Au NPs). Importantly, DNA-Au NPs are universally taken up by cells and can be tailored to target specific intracellular RNA target sequences. Upon encountering a target RNA sequence, Nano-flares turn on a fluorescent signal which can be detected in single cells using fluorescent microscopy, or averaged over millions of cells using fluorescence activated cell sorting (FACS). In contrast to methods which employ the polymerase chain reaction (PCR), cells interrogated using the Nano-flares technology are alive and, thus, available for downstream applications. We propose using the Nano-flare technology to detect and quantify miR and messenger RNA (mRNA) sequences in live prostate cancer cells. Importantly, we will develop a cell model where specific miR and mRNA targets can be manipulated with small molecules in order to evaluate the ability of the Nano-flare system to detect and quantify relative changes in high and low abundance target miR. Results obtained with the Nano-flare system will be directly compared to those obtained using PCR techniques, the current commercial benchmark for detection of these targets. Project success will result in a robust platform for the detection of intracellular miR targets using FACS and confocal fluorescent microscopy. By using qPCR to confirm the Nano-flare results, this project will also set new analytical benchmarks for the real-time detection of miR target sequences inside of live cells, and define what 'low abundance' miR represents in single cells and in ensemble measurements. Finally, completion of this study will bring this enabling technology closer to commercialization and increase the potential for researchers and clinicians, ultimately improving patient health. PUBLIC HEALTH RELEVANCE: Despite the realized and increasing importance of microRNAs in human disease processes, no current methodology exists to detect changes in microRNA levels in live human cells in real-time. Nano-flare technology being developed by AuraSense, LLC will provide this capability, and represents a novel technology that is designed to provide PCR-like phenotypic characterization of intracellular microRNA targets, significantly, in living cells. Successful project completion will provide a unique technological tool for the study of microRNAs to the broader research community, and, ultimately, the clinical community to improve human health.
AuraSense Therapeutics LLC | Date: 2012-09-11
Aspects of the invention relate to novel methods and compositions for assessing the level of cellular uptake of a nanoparticle construct, assessing the level of target binding of a nanoparticle construct and assessing the levels of RNAs and proteins in a given cell.