#3 Nanocubes comprising DNA or RNA core with RNA or DNA hybrid duplex for RNA interference and gene silencing
NIH Title: Nucleic Acid Nanoparticles for Triggering RNA Interference
NIH Reference Number: E-156-2014
Executive Summary:
General Description:
The invention consists of a cube shaped nano scaffold for the delivery of diceable siRNA to target cell types leading to RNAi of target genes. The promise of RNA interference (RNAi) based therapeutics is made evident by the recent surge in formation of companies that are developing methods to deliver functional silencing RNA (siRNA) to target cell types. However, the challenge of developing an effective vehicle for the delivery of stable siRNA to induce RNAi of therapeutically relevant genes has yet to be overcome. The technical roadblocks to developing RNAi therapeutics fall into two general classes: optimization of oligonucleotides for RNAi and achieving targeted delivery to specific tissues.
The NIH inventors created and optimized a series of multi-function nucleic acid nano scaffolds to address these technical hurdles. The nano cube scaffolds consist of a DNA or RNA cubic structure decorated with six hybrid DNA:RNA duplexes, where the RNAs consist of one siRNA strand. After the nano cube scaffolds are spontaneously assembled in vitro, a second set of RNA:DNA duplexes carrying the second siRNA strand are combined in solution. The “split” siRNA strands re-associate to form a diceable RNA duplex. Thus, a single nano cube scaffold can be used to silence six individual genes. The nano cube scaffolds are also highly modular in nature. By incorporating additional nucleic acid structures, such as aptamers, nano cubic scaffolds can be targeted to cell surface proteins of therapeutic relevance.
Scientific Progress:
The NIH researchers have achieved several important development milestones. First, the inventors used computational methods to optimize the nano cube particle structure. Both DNA and RNA nano particles designed in silico can spontaneously self assemble using in vitro transcription as demonstrated by employing a combination of gel based assays, electron microscopy and dynamic light scattering. Formation of functional siRNAs after re-association of the nano cube with the RNA:DNA hybrid duplexes has also been verified using FRET and gel assays.
The inventors have used the nano particles encapsulated within liposomes to induce RNAi in cell culture. For example, nano scaffolds containing “split” siRNAs targeted against six genes required for HIV-1 replication causes significant inhibition of viral protein production. Importantly, DNA-core nano cube scaffolds avoid significant induction of inflammatory cytokines associated with RNA sensing innate immune receptors, a key concern for siRNA based therapies.
Future Direction:
Strength:
Weakness:
Patent Status:
US Application No. 61/989,520
PCT Application No. PCT/US2015/029553
Filing in process
Publications:
Afonin KA et al. PMID: 24189588
Afonin KA et al. PMID: 20802494
Inventor Bio:
Dr. Shapiro received his Ph.D. in computer science from the University of Maryland in 1978, with undergraduate work in mathematics and physics. During his association with the NIH, Dr. Shapiro has done extensive work in image processing, nucleic acid structure prediction and analysis, and computational and experimental nanobiology, leading to several novel algorithms, computer systems, experimental techniques and discoveries in RNA biology. His latest interests include RNA nanobiology, understanding the relationships between RNA structure and function, and the use of parallel high performance computer architectures to solve problems related to RNA computational and experimental biology and molecular modeling.
NIH Reference Number: E-156-2014
Executive Summary:
- Invention Type: Therapeutic
- Patent Status: Patent pending
- Link: https://www.ott.nih.gov/technology/e-156-2014
- NIH Institute or Center: National Cancer Institute (NCI)
- Disease Focus: Cancer, viral infection, other infectious diseases
- Basis of Invention: A 3-D nano cube scaffold based strategy for targeted siRNA delivery to induce RNAi
- How it works: The nano cube scaffolds are decorated with siRNA substrates that are diced upon intracellular uptake, leading to RNAi of target genes
- Lead Challenge Inventor: Bruce Shapiro (NCI)
- Inventors: Bruce Shapiro (NCI), Kirill Afonin (NCI), Viard Mathias (NCI)
- Development Stage: Pre-clinical, in silico design and in vitro assay validation data available
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Novelty:
- This technology opens a new route for the development of “smart” nucleic acid based nanoparticles for a wide range of biomedical applications
- Altering the composition of particles controls immune response
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Clinical Applications:
- Targeted drug delivery to treat cancer, HIV, and other diseases
General Description:
The invention consists of a cube shaped nano scaffold for the delivery of diceable siRNA to target cell types leading to RNAi of target genes. The promise of RNA interference (RNAi) based therapeutics is made evident by the recent surge in formation of companies that are developing methods to deliver functional silencing RNA (siRNA) to target cell types. However, the challenge of developing an effective vehicle for the delivery of stable siRNA to induce RNAi of therapeutically relevant genes has yet to be overcome. The technical roadblocks to developing RNAi therapeutics fall into two general classes: optimization of oligonucleotides for RNAi and achieving targeted delivery to specific tissues.
The NIH inventors created and optimized a series of multi-function nucleic acid nano scaffolds to address these technical hurdles. The nano cube scaffolds consist of a DNA or RNA cubic structure decorated with six hybrid DNA:RNA duplexes, where the RNAs consist of one siRNA strand. After the nano cube scaffolds are spontaneously assembled in vitro, a second set of RNA:DNA duplexes carrying the second siRNA strand are combined in solution. The “split” siRNA strands re-associate to form a diceable RNA duplex. Thus, a single nano cube scaffold can be used to silence six individual genes. The nano cube scaffolds are also highly modular in nature. By incorporating additional nucleic acid structures, such as aptamers, nano cubic scaffolds can be targeted to cell surface proteins of therapeutic relevance.
Scientific Progress:
The NIH researchers have achieved several important development milestones. First, the inventors used computational methods to optimize the nano cube particle structure. Both DNA and RNA nano particles designed in silico can spontaneously self assemble using in vitro transcription as demonstrated by employing a combination of gel based assays, electron microscopy and dynamic light scattering. Formation of functional siRNAs after re-association of the nano cube with the RNA:DNA hybrid duplexes has also been verified using FRET and gel assays.
The inventors have used the nano particles encapsulated within liposomes to induce RNAi in cell culture. For example, nano scaffolds containing “split” siRNAs targeted against six genes required for HIV-1 replication causes significant inhibition of viral protein production. Importantly, DNA-core nano cube scaffolds avoid significant induction of inflammatory cytokines associated with RNA sensing innate immune receptors, a key concern for siRNA based therapies.
Future Direction:
- Investigate the toxicity and anti-cancer activity of RNA nano cubes in preclinical animal models
- Clinical research to study the toxicity of RNA nano cubes in human subjects
- Development of RNA nano sensors using this strategy
- Basic research using RNA nano cubes
Strength:
- More detection sensitivity
- Higher silencing efficiency
- Low cytotoxicity
- Multiple functionality
- Multiple targets including cancer, virus, etc
- Controlled immune response
- Visualization application
- Controlled activation
Weakness:
- Early stage with computer modeling and in vitro/cell culture characterization data
- Requires further validation of nano cubes on the in vivo toxicity and the anti-cancer activity
Patent Status:
US Application No. 61/989,520
PCT Application No. PCT/US2015/029553
Filing in process
Publications:
Afonin KA et al. PMID: 24189588
Afonin KA et al. PMID: 20802494
Inventor Bio:
Dr. Shapiro received his Ph.D. in computer science from the University of Maryland in 1978, with undergraduate work in mathematics and physics. During his association with the NIH, Dr. Shapiro has done extensive work in image processing, nucleic acid structure prediction and analysis, and computational and experimental nanobiology, leading to several novel algorithms, computer systems, experimental techniques and discoveries in RNA biology. His latest interests include RNA nanobiology, understanding the relationships between RNA structure and function, and the use of parallel high performance computer architectures to solve problems related to RNA computational and experimental biology and molecular modeling.
