Synthesis of spherical nucleic acids using lipophilic moieties
Inventors
Kang, Richard • Nallagatla, Subbarao • Anderson, Bart • Kandimalla, Ekambar
Assignees
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Publication Number
US-11696954-B2
Publication Date
2023-07-11
Expiration Date
Abstract
Spherical nucleic acids (SNA) carrying self-aggregating oligonucleotides are described herein. Compositions of the SNA include discrete nanostructures that are not aggregated. Related methods are also described.
Core Innovation
The invention describes spherical nucleic acid (SNA) nanostructures in which a core is surrounded by a lipid shell having an inner surface and an outer surface, and an oligonucleotide is linked to a lipophilic moiety. The oligonucleotide is functionalized to the lipid shell through interaction between the lipid shell and at least a portion of the lipophilic moiety, and the oligonucleotide is oriented radially outwards. The lipophilic moiety is structurally defined to include two or more lipophilic groups rather than a single mono-lipophilic anchor.
The lipophilic moiety includes linked segments L1, L2, and L3, an oligoethylene glycol spacer, and an X unit defined as a doubler or trebler, with at least one of n and n1 not 0. L1, L2, and L3 are linkers that are each independently a phosphodiester or phosphorothioate bond or short chain linkage, and the alkyl group is a C6-C30 saturated or unsaturated alkyl group with m equal to 2-3. The doubler and trebler provide a multi-lipophilic architecture for attachment to the lipid shell.
Using multi-lipophilic moieties, including di-stearyl and related di-alkyl/tri-alkyl architectures, enables formation of discrete, non-aggregated SNAs. The described structures support higher oligonucleotide loading while preventing precipitation and inter-SNA aggregation, including with self-aggregating/self-complementary or G-rich oligonucleotides. The document also states preserved activity with multi-lipophilic moieties, including TLR9-like immunostimulatory cytokines and human PBMC cytokine induction such as IL-6, IL-12p70, TNFα, and IFNα.
Claims Coverage
The independent claim clm-00001 covers a spherical nucleic acid nanostructure with a core, a lipid shell, and a radially outward oriented oligonucleotide functionalized to the lipid shell through a multi-lipophilic moiety. The claim includes defined structural features for the moiety, including a doubler or trebler architecture, linkers, an oligoethylene glycol spacer, and C6-C30 alkyl groups with specified values for m, n, and n1. Dependent claims further add specific lipid and oligonucleotide embodiments and a Z-average diameter range.
Radially outward oriented oligonucleotide on an SNA lipid shell
A spherical nucleic acid comprising a core, a lipid shell having an inner surface surrounding the core and an outer surface, and an oligonucleotide linked to a lipophilic moiety wherein the oligonucleotide is functionalized to the lipid shell through interaction between the lipid shell and at least a portion of the lipophilic moiety and the oligonucleotide is oriented radially outwards.
Multi-lipophilic moiety with doubler or trebler architecture
The lipophilic moiety is comprised of two or more lipophilic groups and has the structure -L1-(Spacer)-L2-(Spacer)n1-L3-X-(alkyl group)m, wherein L1, L2, and L3 are each linkers and are independently a phosphodiester or phosphorothioate bond or short chain linkage, the spacer is an oligoethylene glycol spacer, n and n1 are independently 0-3 with at least one of n and n1 not 0, X is a doubler or trebler, the alkyl group is a C6-C30 saturated or unsaturated alkyl group, and m is 2-3.
Specific lipid shell and oligonucleotide embodiments
Embodiments include a lipid shell comprising 1,2-dioleoyl-sn-glycero-3-phophocholine (DOPC) and oligonucleotide selections including single stranded oligonucleotides and immunostimulatory oligonucleotides; the oligonucleotide may be a self-aggregating oligonucleotide with a self-complementary motif.
Size-constrained SNA compositions by Z-average diameter
Nanostructures having a Z-avg diameter of 30 to 1,300 nm.
Overall, the claim coverage is centered on an SNA in which a lipid shell positions a radially outward oriented oligonucleotide via interaction with a structurally defined multi-lipophilic moiety, with defined linker, spacer, and alkyl architecture. Dependent claim coverage narrows lipid shell embodiments, specifies oligonucleotide motif options, and adds a Z-average diameter range.
Stated Advantages
Enables formation of discrete, non-aggregated SNAs.
Supports higher oligonucleotide loading while preventing precipitation and inter-SNA aggregation.
Preserves immunostimulatory activity when using multi-lipophilic moieties.
Documented Applications
Immune response eliciting by immunostimulatory oligonucleotides, including TLR9 stimulation described as TLR9-like immunostimulatory cytokine induction.
Gene regulation contexts are referenced through antisense and inhibitory oligonucleotides as oligonucleotide embodiments within the disclosed SNA compositions.
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