Compositions containing nucleic acid nanoparticles with modular functionality
Inventors
Rushworth, James Luke • Foot, George William • Rosell, Anna Perdrix • Brzosko, Zuzanna Aleksandra
Assignees
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Publication Number
US-11357865-B2
Publication Date
2022-06-14
Expiration Date
Abstract
The invention provides compositions containing cargo molecules attached to elements that improve the function of the cargo molecules in the body of a subject. The compositions are useful for therapeutic and diagnostic purposes. Furthermore, the invention outlines ways in which these compositions can be produced; the core molecule can be functionalized, via bioorthogonal click chemistry, in such a way as to impart modular characteristics. This functionalization simultaneously allows for loading of biologically relevant cargo and provides stabilization to the overall structure of the molecule.
Core Innovation
Embodiments describe a composition comprising a self-assembled RNA nanoparticle having at least four oligonucleotide strands of 3 to 200 nucleotides and a four-sided polygonal tertiary structure formed by four or more junctions. Each junction is formed by at least two of the oligonucleotide strands, and at least one oligonucleotide strand includes one or more reactive sites that allow conjugation.
Cargo molecules are conjugated to the self-assembled RNA nanoparticle at the one or more reactive sites, providing covalent attachment of cargo molecules to the RNA nanoparticle. Covalent bond formation is implemented by reactions selected from CuAAC, SPAAC, RuAAC, IEDDA, SuFEx, SPANC, hydrazone/oxime ether formation, thiol-ene radical reaction, thiol-yne radical reaction, thiol-Michael addition reaction, thiol-isocyanate reaction, thiol-epoxide click reaction, nucleophilic ring opening reactions, and traceless Staudinger ligation.
The composition further supports cargo molecules linked to additional cargo molecules through cleavable or non-cleavable linker types, including thiol-cleavable, hydroxylamine-cleavable, base-cleavable, and Meldrum’s acid derivative cleavable linkers. The document also describes modular cargo architectures, including polymeric cargo linkage, and self-assembled RNA nanoparticle constructs with cargo-dependent nanoparticle construction.
Claims Coverage
The independent claims are directed to a self-assembled RNA nanoparticle composition with covalent cargo conjugation via selected reaction classes. Across the claims, the coverage centers on a four-sided polygonal tertiary structure, strand-based reactive sites, and cargo-linking architectures, with six inventive features identified.
Self-assembled RNA nanoparticle with covalent cargo conjugation at reactive sites
A composition comprising a self-assembled RNA nanoparticle having at least four oligonucleotide strands of 3 to 200 nucleotides, wherein at least one oligonucleotide strand is functionalized with one or more reactive sites that allow formation of a covalent bond via reactions selected from CuAAC, SPAAC, RuAAC, IEDDA, SuFEx, SPANC, hydrazone/oxime ether formation, thiol-ene radical reaction, thiol-yne radical reaction, thiol-Michael addition reaction, thiol-isocyanate reaction, thiol-epoxide click reaction, nucleophilic ring opening reactions, and traceless Staudinger ligation, and wherein one or more cargo molecules are conjugated to the self-assembled RNA nanoparticle at the one or more reactive sites.
Four-sided polygonal tertiary structure from junctions formed by oligonucleotide strands
The self-assembled RNA nanoparticle includes a four-sided polygonal tertiary structure of four or more junctions, each junction formed by at least two of the oligonucleotide strands.
Cargo linked through cleavable linker types
At least one of the first and second cargo molecules is linked to a third cargo molecule via a cleavable linker type, including thiol-cleavable linkers, hydroxylamine-cleavable linker, base-cleavable linker, and a Meldrum’s acid derivative.
Polymeric cargo linkage architecture
The second cargo molecule is linked to any number of cargo molecules in a polymeric fashion.
Biological activities attributable to the RNA nanoparticle
The RNA nanoparticle composition can perform one or more biological activities including binding to serum proteins or receptors, promoting endosomal escape, tissue targeting, modulating biodistribution, inducing or preventing immunological responses, enhancing cellular uptake, modulating gene expression, inducing cytotoxicity, and/or having a therapeutic effect, or combinations thereof.
Attachment reaction characteristics for RNA nanoparticle to cargo
Attaching the RNA nanoparticle to at least one cargo molecule can be achieved via a reaction having one or more specified features including one-pot operation, water-undeveloped conditions, minimal byproduct formation, and a high thermodynamic driving force yielding a single reaction product.
Claim coverage centers on the RNA nanoparticle composition featuring a four-sided polygonal tertiary structure and reactive-site functionalization enabling covalent cargo conjugation through selected reaction chemistries, with additional scope for cleavable linkers, polymeric cargo architectures, reaction characteristics, and enumerated biological activities.
Stated Advantages
Allows covalent bond formation between cargo molecules and the self-assembled RNA nanoparticle at reactive sites using a selected set of covalent reaction classes.
Documented Applications
RNA nanoparticle compositions for biological activities including binding to serum proteins or receptors, promoting endosomal escape, tissue targeting, modulating biodistribution, inducing or preventing immunological responses, enhancing cellular uptake, modulating gene expression, inducing cytotoxicity, and/or having a therapeutic effect, or combinations thereof.
Knockdown of polo-like kinase 1 (PLK1) in MDA-MB-231 cells using conjugated siRNA and nanoparticle constructs.
Combinatorial dual-siRNA cargo strategies using disulfide and TTTT spacer architectures showing higher knockdown outcomes.
Dual conjugation approaches using GalNAc and cholesterol cargo strategies.
Aptamer binding and uptake assays using EGFR-targeting aptamers, including binding and uptake analyses in the described context.
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