Multifunctional RNA nanoparticles and methods of use
Inventors
Shapiro, Bruce A. • Afonin, Kirill A. • Viard, Mathias D. • Martins, Angelica N.
Assignees
US Department of Health and Human Services
Publication Number
US-10301621-B2
Publication Date
2019-05-28
Expiration Date
2034-09-17
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Abstract
The instant invention provides RNA nanoparticles and R/DNA chimeric nanoparticles comprising one or more functionalities. The multifunctional RNA nanoparticles are suitable for therapeutic or diagnostic use in a number of diseases or disorders.
Core Innovation
The invention provides polyvalent RNA nanoparticles and R/DNA chimeric nanoparticles comprising one or more functionalities, such as inhibitory nucleic acids, fluorescent dyes, small molecules, RNA-DNA hybrids with split functionalities, proteins, therapeutic agents, and imaging agents. These multifunctional RNA nanoparticles can assemble into higher order structures including rings, cages, and nanotubes, and they are suitable for therapeutic or diagnostic use in a wide range of diseases or disorders.
The problem being addressed involves the challenges in delivering short interfering RNAs (siRNAs) specifically and effectively into tumor or target cells after systemic administration. Current issues in RNA interference (RNAi) therapeutics include transport, targeting, stabilization of siRNAs, and avoiding immune responses. The invention seeks to provide RNA nanoscaffolds that enhance cell-targeting, ease of synthesis, triggered activation of therapeutic functionalities, and safe, efficient delivery of therapeutic and diagnostic siRNAs.
Claims Coverage
The patent includes 19 inventive features primarily directed to RNA nanoparticles or R/DNA chimeric nanoparticles with nanoring structures functionalized via toehold interactions and related compositions, methods, and applications.
RNA nanoparticle or R/DNA chimeric nanoparticle with toehold interactions
An RNA nanoparticle (RNA NP) or R/DNA chimeric nanoparticle (R/DNA NP) comprising a nanoring with one or more functionalities attached by toehold interactions where each toehold is a single strand nucleation site hybridizing to a complementary nucleic acid on the functionalities. The functionalities include inhibitory nucleic acids, fluorescent dyes, small molecules, RNA-DNA hybrids with split functionalities, split lipase, split GFP, proteins, targeting moieties, therapeutic agents, or imaging agents.
Functionalities comprising RNA-DNA hybrid or double strand RNA arm extensions
The nanoparticle functionalities comprise one or more RNA-DNA hybrid arm extensions or double strand RNA arm extensions, or a combination thereof attached to the nanoring via toehold interactions.
Triggered release of inhibitory nucleic acid from RNA-DNA hybrid arm extensions
One or more RNA-DNA hybrid arm extensions is capable of triggered release, formation and/or activation of an inhibitory nucleic acid.
Inhibitory nucleic acids as siRNAs, aptamers, and ribozymes
The inhibitory nucleic acids functionalized on the nanoparticle are selected from siRNAs, RNA or DNA aptamers, and ribozymes.
Uniform functionalities on nanoparticles
The one or more functionalities attached to the nanoparticle can be the same.
Diverse functionalities on nanoparticles
The one or more functionalities attached to the nanoparticle can be different.
Triggered activation via complementary chimeric nanoparticles
The nanoparticle is capable of triggered release, formation and/or activation of inhibitory nucleic acids in the presence of a second and complementary chimeric nanoparticle(s).
Complementary RNA-DNA hybrid in second chimeric nanoparticle
The second chimeric nanoparticle comprises a second RNA-DNA hybrid whose sequences are complementary to the RNA-DNA hybrid arm extension of the nanoring.
Complementary DNA toehold sequences facilitate reassociation and siRNA formation
The RNA-DNA hybrid arm extensions of the nanoring and complementary RNA-DNA hybrid each include complementary DNA toehold sequences that enable reassociation and formation of active siRNA from the complementary RNA sequences.
siRNA inhibition of target RNA
The siRNA formed from reassociation inhibits a target RNA.
Target RNA encodes apoptosis inhibitor protein
The target RNA inhibited by siRNA encodes an apoptosis inhibitor protein.
Target RNA is pathogenic RNA genome or transcript
The target RNA is a pathogenic RNA genome, an RNA transcript derived from the pathogenic genome, or a portion thereof.
Pathogenic agents include virus, bacteria, fungus, or parasite
The pathogenic agent corresponding to the target RNA can be a virus, bacteria, fungus, or parasite.
Target RNA is viral RNA genome or portion thereof
The target RNA is a viral RNA genome or a portion thereof.
Pharmaceutical composition comprising functional RNA nanoparticles
A pharmaceutical composition comprising any RNA NP or R/DNA NP with functionalities as described in the invention.
Pharmaceutical composition with pharmaceutically acceptable excipient
The pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, or diluent.
Kit comprising functional RNA nanoparticles and instructions
A kit comprising the RNA NP or R/DNA NP as described and a set of instructions for use.
Target RNA includes one or more HIV-1 transcripts
The target RNA includes one or more HIV-1 transcripts.
HIV-1 transcripts targeted include PBS-matrix, capsid, protease, reverse transcriptase, envelope, Nef, and Rev-Tat
The HIV-1 transcripts targeted by siRNAs on the nanoparticle are selected from transcripts encoding primer binding site (PBS)-matrix, capsid, protease, reverse transcriptase, envelope, Nef, and Rev-Tat.
The claims cover multifunctional RNA and R/DNA chimeric nanoparticles with nanoring structures functionalized through toehold interactions to deliver various agents including inhibitory nucleic acids and targeting moieties, with methods to activate functionalities conditionally inside cells, pharmaceutical compositions containing such nanoparticles, kits including them, and target RNA molecules including HIV-1 transcripts.
Stated Advantages
RNA nanoparticles provide a nanoparticle effect that improves cellular uptake and specific gene silencing at low concentrations in vitro and in vivo.
Nanoparticles offer increased protection against ribonuclease degradation while accommodating additional functional groups like aptamers for cellular targeting.
Use of natural RNA nanoscaffolds offers biocompatibility and inherent ability to self-assemble and spatially arrange multiple functionalities.
Nanoparticles composed of RNA do not induce significant immune responses unlike protein nanoparticles.
Nanoparticles are smaller than many currently available nanoparticles, allowing increased efficiency of administration.
Nanorings functionalized with siRNAs demonstrate higher detection sensitivity and increased silencing efficiency compared to free siRNAs.
Triggerable activation of therapeutic functionalities is possible via complementary RNA/DNA hybrid reassociation, providing control over when the therapeutic becomes active.
Documented Applications
Therapeutic or diagnostic use of multifunctional RNA nanoparticles and R/DNA chimeric nanoparticles in a number of diseases including cancers and viral infections.
Combinatorial RNA interference therapy using nanorings functionalized with multiple siRNAs targeting HIV-1 genes for simultaneous gene silencing.
Targeted delivery of therapeutics to cells expressing specific receptors, exemplified by RNA aptamer functionalization targeting human Epidermal Growth Factor Receptor (EGFR) on cancer cells.
Triggered intracellular activation of therapeutic siRNAs from inactive RNA-DNA hybrids targeting disease-related genes.
Pharmaceutical compositions and kits containing RNA nanoparticles functionalized with therapeutic, diagnostic, or imaging agents for use in treatment or prevention of diseases.
Methods of inhibiting gene expression, killing pathogen-infected cells, inhibiting replication of pathogens, treating pathogenic infections, and treating neoplasia by administering the RNA or R/DNA nanoparticles.
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