Triggering RNA interference with RNA-DNA and DNA-RNA nanoparticles
Inventors
Shapiro, Bruce A. • Afonin, Kirill A. • Viard, Mathias D.
Assignees
US Department of Health and Human Services
Publication Number
US-10517890-B2
Publication Date
2019-12-31
Expiration Date
2035-05-06
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Abstract
The instant invention provides RNA nanocubes, DNA nanocubes and R/DNA chimeric nanocubes comprising one or more functionalities. The multifunctional RNA nanocubes are suitable for therapeutic or diagnostic use in a number of diseases or disorders.
Core Innovation
The invention provides RNA nanocubes, DNA nanocubes, and RNA/DNA chimeric nanocubes that comprise one or more functionalities. These nanocubes are formed from multiple strands of RNA or DNA oligonucleotides that self-assemble into cube-like three-dimensional structures. The nanocubes include single-stranded RNA or DNA arms capable of annealing to cognate complementary strands, forming RNA-DNA hybrid duplexes. The multifunctional RNA nanocubes are suitable for therapeutic or diagnostic use in a wide range of diseases or disorders.
The core innovation centers around the conditional activation of functionalities such as Dicer substrate RNAs or RNA aptamers that are initially split into two RNA-DNA hybrids. One hybrid is associated with the nanocube arm, and the other is a free complementary RNA-DNA hybrid molecule that is inactive in its hybrid state. These hybrids contain single-stranded DNA toeholds that are complementary and enable interaction and reassociation when present in close proximity, leading to strand swapping that forms RNA-RNA or DNA-DNA duplexes. This strand exchange releases active functionalities, such as active double-stranded RNA capable of triggering RNA interference (RNAi).
The problem addressed by the invention arises from challenges in RNAi therapeutics, including transporting, targeting, and stabilizing siRNAs within tumor cells after systemic administration. Although various nanoparticles have been used, delivery specificity, stability, and triggered activation of therapeutic functionalities remain inadequately solved. The invention addresses these challenges by providing nanocube-based siRNA scaffolds that offer reduced immunogenicity, enhanced stability, programmable assembly, and controlled activation of RNAi via reassociation with cognate hybrids.
Claims Coverage
The patent claims several independent inventions relating to DNA and RNA nanoparticles capable of conditional activation for RNA interference (RNAi) through the use of RNA-DNA (R/DNA) hybrid arms and cognate hybrid molecules. The main inventive features focus on the structure, functional activation mechanism, and pharmaceutical compositions and methods involving these nanoparticles.
DNA nanoparticles capable of conditional RNAi activation
A six-stranded DNA oligonucleotide nanocube with three single-stranded thymine residues at each corner, where one to six DNA strands have covalently attached single-stranded R/DNA arms. Cognate single-stranded R/DNA molecules anneal to these arms forming R/DNA hybrid arms. The DNA nanocube becomes RNAi-activated upon association with free R/DNA hybrid molecules that enable strand displacement, forming RNA-RNA or DNA-DNA duplex arms and releasing corresponding free hybrids.
RNA nanoparticles capable of conditional RNAi activation
A six-stranded RNA oligonucleotide nanocube with three single-stranded uracil residues at each corner, where one to six RNA strands have covalently attached single-stranded R/DNA arms. Cognate single-stranded R/DNA molecules anneal to these arms forming R/DNA hybrid arms. RNA nanocubes become RNAi-activated upon association with free R/DNA hybrid molecules that facilitate strand displacement to produce RNA-RNA or DNA-DNA duplex arms and free duplex molecules.
Pharmaceutical compositions containing inactive DNA or RNA nanocubes activatable for RNAi
Compositions comprising DNA or RNA nanocubes with R/DNA hybrid arms and pharmaceutical excipients, wherein the nanocubes are inactive until combined with free cognate R/DNA hybrid molecules that trigger reassociation to form active RNAi-inducing duplex arms.
Methods of treating diseases using compositions comprising activatable DNA or RNA nanocubes
Methods of treating subjects having diseases or disorders treatable with RNAi agents by administering compositions containing the DNA or RNA nanocubes described, which become activated to induce RNA interference.
Use of R/DNA hybrid arms for conditional activation of RNAi nanoparticles
Incorporation of single-stranded R/DNA arms in DNA or RNA nanocube scaffolds that are complementary to free R/DNA hybrid molecules, allowing controlled triggering of RNA interference activity via strand displacement and reassociation.
Structural features of DNA and RNA nanocubes
Nanocubes formed from six oligonucleotides each having sequence structures with single-stranded thymine or uracil residues at corners and covalently attached single-stranded R/DNA arms at the 3′ ends, allowing self-assembly and functionalization with split RNAi agents such as siRNAs or DsiRNAs.
The claims broadly cover DNA and RNA six-stranded nanocubes functionalized with R/DNA hybrid arms that are activatable for RNA interference upon association with complementary free R/DNA hybrids. The inventions encompass the nanoparticle structures themselves, pharmaceutical compositions including these nanoparticles, and methods for treating diseases by utilizing these activatable nanoparticles. Key inventive aspects include the structural design of the nanocubes, the use of single-stranded DNA toeholds for conditional activation through strand displacement, and the therapeutic application of this technology.
Stated Advantages
Reduced immunogenicity compared to protein nanoparticles and non-cube RNA nanoparticles, especially notable for DNA nanocubes.
Enhanced stability of the nanocube structures, increasing resistance to degradation such as by human blood serum nucleases.
Capability to form initially inactive particles that are activated for RNAi activity only upon interaction with complementary RNA-DNA hybrids, enabling spatial and temporal control over therapeutic activation.
Precise control over stoichiometry and composition of RNA interference agents loaded on the nanocubes.
Improved visualization and labeling possibilities (e.g., fluorescent labeling) particularly in DNA nanocubes.
Potential for combinatorial therapy by simultaneous delivery of multiple siRNAs targeting different gene sequences.
Documented Applications
Therapeutic and diagnostic uses of RNA and DNA nanocubes functionalized with RNA interference agents for treatment of diseases or disorders.
Combinatorial RNAi therapy targeting multiple viral genes such as those in HIV-1 to reduce viral infectivity and production.
Treatment of pathological conditions including cancer, viral infections, and other diseases amenable to RNA interference methods.
Use as drug delivery vehicles providing controlled, targeted and stable delivery of nucleic acid-based therapeutics.
Use of nanocubes as scaffolds for diverse functionalities including imaging agents, aptamers, fluorescent dyes, and proteins.
Biomedical applications employing activation of split RNA-DNA functionalities within cells for controlled therapeutic intervention.
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