Lipid nanoparticles spherical nucleic acid

Abstract

To treat disease, DNA and RNA therapeutics need to be delivered to target tissues and provide sustained benefits without side effects. Lipid nanoparticle spherical nucleic acids address this unmet need by using DNA and RNA sequences for nanoparticle targeting and tissue specificity. This lipid SNA structure has significantly different biodistribution properties than both lipid particles (loaded with nucleic acid) or traditional SNAs (liposomes and gold cores).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 136,501, filed January 12, 2021, which is incorporated by reference in its entirety herein.

[0002] Government Interest Statement This invention was made with Government support under Grant Nos. CA208783, CA221747, and CA199091 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Submitted Materials A sequence listing, which is part of this disclosure, is submitted as a text file at the same time as the specification. The name of the text file containing the sequence listing is "2021-002_Seqlisting.txt", which was created on January 10, 2022, and is 643 bytes in size. The contents of the sequence listing are incorporated herein by reference. [Background technology]

[0004] Nucleic acids (DNA and RNA) have many potential applications in both therapy and diagnosis, but effective delivery at clinically relevant doses remains a challenge. The structure of these molecules creates limitations to their delivery. These limitations include rapid degradation by nucleases, poor biodistribution properties, low accumulation in target tissues, and sequestration within cellular compartments. To address these issues, many different nanoparticle carrier structures for nucleic acids have been explored.

[0005] Lipid nanoparticles are useful for facilitating intracellular delivery and cytoplasmic recognition of oligonucleotides, as well as expression of messenger RNA. However, their ability to target specific tissues appears to depend on endogenous lipid transport pathways (see, e.g., Akinc et al., Molecular Therapy (2010) 18: 1357-1364). To address this limitation, extensive screening of lipid structures or sterols is often required [see, e.g., Love et al., Proceedings of the National Academy of Sciences of the United States of America (2010) 107: 1864-1869, and Patel et al., Nature Communications (2020) 983]. Although these studies have revealed some relationships between nanoparticle structures and their distribution properties, there remains a need to develop systems with predictable nanoparticle targeting capabilities. Summary of the Invention

[0006] Many diseases can be treated with nucleic acid (DNA or RNA) based therapeutics that silence harmful genes, replace defective genes, and edit mutations in the patient's DNA. However, in the body, the active sequences often do not reach the target tissues and cells. Lipid nanoparticles (LNPs) are some of the most effective carriers of nucleic acids. LNP carriers are effective for delivery to easily accessible targets, but often require screening thousands of different carrier structures to find significant enhancement in cell populations outside the bloodstream and liver. Spherical nucleic acid (SNA) structures facilitate this approach by using short DNA sequences on the surface of existing nanoparticle structures to target their delivery. The radially oriented outer sequences change both the destination of the nanoparticles in the body and their activity. The outer DNA sequences target the delivery of the associated LNPs encapsulating the nucleic acids used for gene silencing, gene replacement, or gene editing. The outer DNA sequences provide the "address" to which the LNPs are delivered. Because combinations of DNA sequences can form many different structures, this strategy offers numerous options for adding targeting, stimuli-responsiveness, and diagnostic capabilities to the LNP structure.

[0007] Applications of the technology described herein include, but are not limited to: Delivery of RNA to facilitate expression of therapeutic proteins Delivery of small interfering RNA (siRNA) for therapeutic gene silencing Delivery of multiple DNA and RNA strands for genome editing Delivery of DNA and RNA probes for diagnostics Delivery of DNA and RNA strands designed to modulate the immune system Co-delivery of DNA and RNA therapeutics with other medicines Targeted delivery of encapsulated drugs

[0008] Advantages of the technology described herein include, but are not limited to: Lipid nanoparticle (LNP) structures exist that deliver different DNA and RNA sequences -Delivery to target tissues without side effects is a central challenge in this field. There are two main approaches to targeting LNPs: 1.) synthesis of novel lipid structures and LNP compositions, and 2.) decoration of the LNP surface with targeting peptides and antibodies. Lipid nanoparticle spherical nucleic acid (LNP-SNA) decorates the surface of LNPs with DNA and / or RNA sequences DNA and RNA sequences on the surface of LNP-SNAs enhance the function and alter the tissue targeting of nanoparticles. This represents a highly modular platform because DNA and RNA can form many different structures and, in theory, can be designed to bind any target with high specificity. DNA and RNA are useful as sensors and can be used to design therapeutic and diagnostic (theranostic) structures from lipid nanoparticles.

[0009] Thus, in some aspects, the present disclosure provides lipid nanoparticle spherical nucleic acid (LNP-SNA) comprising a lipid nanoparticle core and an oligonucleotide shell comprised of oligonucleotides bound to the exterior of the lipid nanoparticle core, the lipid nanoparticle core comprising encapsulated oligonucleotides, ionizable lipids, phospholipids, sterols, and lipid-polyethylene glycol (lipid-PEG) conjugates, wherein at least 10% of the oligonucleotides in the oligonucleotide shell are covalently bound to the exterior of the lipid nanoparticle core via the lipid-PEG conjugates. In various embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the oligonucleotides in the oligonucleotide shell are covalently bound to the exterior of the lipid nanoparticle core via the lipid-PEG conjugates. In some embodiments, 100% of the oligonucleotides in the oligonucleotide shell are covalently bound to the exterior of the lipid nanoparticle core via the lipid-PEG conjugates. In some embodiments, the oligonucleotide shell comprises about 5 to about 1000 oligonucleotides. In some embodiments, the oligonucleotide shell comprises about 100 to about 1000 oligonucleotides. In further embodiments, the oligonucleotide shell comprises about 400 oligonucleotides. In various embodiments, each oligonucleotide in the oligonucleotide shell is about 5 to about 100 nucleotides in length. In further embodiments, each oligonucleotide in the oligonucleotide shell is about 10 to about 50 nucleotides in length. In some embodiments, each oligonucleotide in the oligonucleotide shell is about 25 nucleotides in length. In some embodiments, each oligonucleotide in the oligonucleotide shell has the same nucleotide sequence. In some embodiments, the oligonucleotide shell comprises at least two oligonucleotides having different nucleotide sequences. In various embodiments, the oligonucleotide shell is comprised of single-stranded, double-stranded DNA oligonucleotides, or combinations thereof.In further embodiments, the oligonucleotide shell is comprised of single stranded, double stranded RNA oligonucleotides, or combinations thereof. In some embodiments, the oligonucleotide shell is comprised of single stranded DNA oligonucleotides, double stranded DNA oligonucleotides, single stranded RNA oligonucleotides, double stranded RNA oligonucleotides, or combinations thereof. In some embodiments, at least one oligonucleotide in the oligonucleotide shell is a targeting oligonucleotide. In further embodiments, each oligonucleotide in the oligonucleotide shell is a targeting oligonucleotide. In some embodiments, at least one oligonucleotide in the oligonucleotide shell is (GGT). n In some embodiments, each oligonucleotide within the shell of oligonucleotides comprises or consists of the nucleotide sequence: (GGT) nThe oligonucleotide shell comprises or consists of a nucleotide sequence, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. In some embodiments, n is 7. In some embodiments, at least one oligonucleotide in the shell of the oligonucleotide is an aptamer. In some embodiments, at least one oligonucleotide in the shell of the oligonucleotide comprises a detectable marker. In further embodiments, the shell of the oligonucleotide comprises an inhibitory oligonucleotide, an immunostimulatory oligonucleotide, a gene editor substrate DNA or RNA, or a combination thereof. In still further embodiments, the inhibitory oligonucleotide is an antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme. In some embodiments, the immunostimulatory oligonucleotide is a CpG motif-containing oligonucleotide, a double-stranded DNA oligonucleotide, a double-stranded RNA oligonucleotide, or a single-stranded RNA oligonucleotide. In some embodiments, the encapsulated oligonucleotide is comprised of DNA, RNA, or a combination thereof. In further embodiments, the encapsulated oligonucleotide is an inhibitory oligonucleotide, an mRNA, an immunostimulatory oligonucleotide, an mRNA encoding a gene editor protein, or a DNA or RNA gene editor substrate. In some embodiments, the inhibitory oligonucleotide is an antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme. In further embodiments, the immunostimulatory oligonucleotide is a CpG motif-containing oligonucleotide. In further embodiments, the immunostimulatory oligonucleotide is a CpG motif-containing oligonucleotide, a double-stranded DNA (dsDNA), a double-stranded RNA, or a single-stranded RNA (ssRNA). In various embodiments, the encapsulated oligonucleotide is about 5 to about 5000 nucleotides in length.In further embodiments, the encapsulated oligonucleotide is about 10 to about 4500 nucleotides in length. In yet further embodiments, the encapsulated oligonucleotide is about 1500 nucleotides in length. In some embodiments, the lipid nanoparticle core comprises a plurality of encapsulated oligonucleotides. In some embodiments, at least one oligonucleotide in the plurality of encapsulated oligonucleotides comprises a detectable marker. In various embodiments, the plurality of encapsulated oligonucleotides comprises an inhibitory oligonucleotide, an mRNA, an immunostimulatory oligonucleotide, an mRNA encoding a gene editor protein, a DNA or RNA gene editor substrate, or combinations thereof. In further embodiments, the inhibitory oligonucleotide is an antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme. In further embodiments, the immunostimulatory oligonucleotide is a CpG motif-containing oligonucleotide, a double-stranded DNA (dsDNA), a double-stranded RNA, or a single-stranded RNA (ssRNA). In some embodiments, each oligonucleotide in the plurality of encapsulated oligonucleotides is about 10 to about 50 nucleotides in length. In further embodiments, each oligonucleotide in the plurality of encapsulated oligonucleotides is about 50 nucleotides in length. In some embodiments, each oligonucleotide in the plurality of encapsulated oligonucleotides has the same nucleotide sequence. In some embodiments, the plurality of encapsulated oligonucleotides comprises at least two oligonucleotides having different nucleotide sequences. In some embodiments, the ionizable lipid is dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), C12-200, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), or a combination thereof. In some embodiments, the ionizable lipid is dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA).In some embodiments, the LNP-SNA comprises a molar fraction of ionizable lipids that is about 50% of the total lipids in the LNP-SNA. In further embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dihexadecanoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or a combination thereof. In some embodiments, the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In further embodiments, the LNP-SNA comprises a molar fraction of phospholipids that is about 1% to about 25% of the total lipids in the LNP-SNA. In some embodiments, the LNP-SNA comprises a molar fraction of phospholipids that is 3.5% or about 3.5% of the total lipids in the LNP-SNA. In some embodiments, the sterol is 3β-hydroxycholest-5-ene (cholesterol), 9,10-secocholesta-5,7,10(19)-trien-3β-ol (vitamin D3), 9,10-secoegosta-5,7,10(19),22-tetraen-3β-ol (vitamin D2), calcipotriol, 24-ethyl-5,22-cholestadien-3β-ol (stigmasterol), 22,23-dihydrostigmasterol (β-sitosterol), 3,28-dihydroxy-lupeol ( betulin), lupeol, ursolic acid, oleanolic acid, 24α-methylcholesterol (campesterol), 24-ethylcholesta-5,24(28)E-dien-3β-ol (fucosterol), 24-methylcholesta-5,22-dien-3β-ol (brassicasterol), 24-methylcholesta-5,7,22-trien-3β-ol (ergosterol), 9,11-dehydroergosterol, daucosterol, or any of the foregoing sterols modified with one or more amino acids. In some embodiments, the LNP-SNA comprises a molar fraction of sterols that is about 25% to about 45% of the total lipids in the LNP-SNA.In some embodiments, the LNP-SNA comprises a molar fraction of sterol that is 45% or about 45% of the total lipids in the LNP-SNA. In further embodiments, the sterol is cholesterol. In yet further embodiments, the LNP-SNA comprises a molar fraction of cholesterol that is 45% or about 45% of the total lipids in the LNP-SNA. In some embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate comprises 2000 Dalton (Da) polyethylene glycol. In some embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate is lipid-PEG-maleimide. In further embodiments, the lipid-PEG-maleimide is 1,2-dipalmitryl-sn-glycero-3-phosphoethanolamine (DPPE) conjugated to a 2000 Da polyethylene glycol maleimide, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) conjugated to a 2000 Da polyethylene glycol maleimide, or a combination thereof. In some embodiments, the LNP-SNA comprises a molar fraction of lipid-PEG conjugate that is about 1.5% to about 3.5% of the total lipid in the LNP-SNA. In some embodiments, the LNP-SNA comprises a molar fraction of lipid-PEG conjugate that is 1.5% or is about 1.5% of the total lipid in the LNP-SNA. In various embodiments, the mass ratio between the ionizable lipid and the encapsulated oligonucleotide is about 20:1 to about 5:1. In some embodiments, the LNP-SNAs of the present disclosure further comprise a therapeutic agent encapsulated in the lipid nanoparticle core. In further embodiments, the LNP-SNAs of the present disclosure comprise a therapeutic agent bound to the exterior of the lipid nanoparticle core. In further embodiments, the therapeutic agent is an antibody or antibody fragment, a small molecule, a peptide, an antibiotic, a growth factor, a hormone, an interferon, an interleukin, an antifungal agent, an antiviral agent, a chemotherapeutic agent, or a combination thereof. In some embodiments, the LNP-SNA of the present disclosure further comprises a targeting peptide, a targeting antibody, or a combination thereof attached to the exterior of the lipid nanoparticle core.

[0010] In some aspects, the present disclosure provides a composition comprising a plurality of lipid nanoparticle spherical nucleic acids (LNP-SNAs) of the present disclosure. In some embodiments, the composition of the present disclosure further comprises a therapeutic agent.

[0011] In some aspects, the present disclosure provides a method of inhibiting expression of a gene comprising hybridizing a polynucleotide encoding a gene product with a lipid nanoparticle spherical nucleic acid (LNP-SNA) or composition of the present disclosure, wherein hybridization between the polynucleotide and one or more oligonucleotides within the shell of the oligonucleotide occurs over the length of the polynucleotide with a sufficient degree of complementarity to inhibit expression of the gene product. In some embodiments, expression of the gene product is inhibited in vivo. In some embodiments, expression of the gene product is inhibited in vitro.

[0012] In some aspects, the present disclosure provides a method for upregulating the activity of a Toll-like receptor (TLR), comprising contacting a cell having a toll-like receptor with a lipid nanoparticle spherical nucleic acid (LNP-SNA) or composition of the present disclosure. In some embodiments, the shell of the oligonucleotide comprises one or more oligonucleotides that are TLR agonists. In further embodiments, the toll-like receptor is toll-like receptor 1, toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 11, toll-like receptor 12, toll-like receptor 13, or a combination thereof. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo.

[0013] In some aspects, the present disclosure provides a method for downregulating the activity of a Toll-like receptor (TLR), comprising contacting a cell having a toll-like receptor with a lipid nanoparticle spherical nucleic acid (LNP-SNA) or composition of the present disclosure. In some embodiments, the shell of the oligonucleotide comprises one or more oligonucleotides that are TLR antagonists. In further embodiments, the toll-like receptor is toll-like receptor 1, toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 11, toll-like receptor 12, toll-like receptor 13, or a combination thereof. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo.

[0014] In some aspects, the present disclosure provides a method of treating a disorder, comprising administering to a subject in need thereof an effective amount of a lipid nanoparticle spherical nucleic acid (LNP-SNA) or composition of the present disclosure, wherein administering treats the disorder. In some embodiments, the disorder is cancer, an infectious disease, an autoimmune disease, or a combination thereof. [Brief description of the drawings]

[0015] [Figure 1] The synthesis of LNP-SNA is shown. Nucleic acid-loaded LNPs are formed by the ethanol dilution method. DNA or RNA dissolved in 10 mM sodium citrate buffer at pH 4.0 is mixed with lipids and cholesterol in ethanol at a volume ratio of 3:1. Then, LNPs containing lipid-PEG-maleimide (red circle) are mixed with 3'-SH DNA (blue) at room temperature for 2 h, resulting in LNP-SNA. [Diagram 2]Characterization of LNP-SNAs with encapsulated mRNA. (A) Plot of the average of three NanoSight runs of an LNP-SNA composed of 3.5% DOPE, 45% cholesterol, 50% D-Lin-MC3-DMa, and 1.5% DMPE-PEG(2000)-maleimide with encapsulated Luc2 mRNA. (B) Cryo-TEM image of the same SNA. [Diagram 3] Figure 1 shows the conjugation of DNA to LNP and the formation of LNP-SNA confirmed. A 1% agarose gel run in TAE buffer confirmed the conjugation of T21 DNA to LNP after shaking for 2 hours at room temperature. One equivalent of Cy5.5-labeled T21-SH DNA sequence was added to a formulation containing 3.5 mol% C14-PEG(2000)-maleimide. The presence of bands at higher MW than the free Cy5.5 DNA (lane 1) indicated that they were conjugated to lipid-PEG. [Figure 4] The activity of LNP-SNA in cell assays is shown. (A) IRF3 induction of B-33LNP-SNA, 2'3'-cGAMP, and B-33LNP-SNA mixed with free dsDNA in Raw264.7 cell line (red ribbons represent 95% CI, n=3 biologically independent replicates). (B) B-35SNA silences Luc2 expression in U87-MG-Luc2 cell line compared to B-35 formulation with encapsulated siGFP control (24 hours, normalized to cell viability using CellTiter-Fluor™ assay, n=3 biologically independent replicates). (C) B-35SNA enhances Luc2 gene silencing compared to a comparable LNP formulation with no DNA conjugated to its surface (24 h, normalized to untreated cell luminescence, one-tailed Student's t test, *=p<0.05, **=p<0.01, n=3 biologically independent replicates). [Diagram 5]Figure 1 shows mRNA delivery to major organs using LNP-SNA in C57BL / 6 mice. LNP-SNA enhanced hepatic mRNA expression compared to the equivalent LNP. Luminescence was detected in harvested organs 6 hours after administration of 0.1 mg kg-1 Luc mRNA via the lateral tail vein. LNP-SNA demonstrated organ-specific functionality with respect to mRNA expression. Luminescence was detected in harvested organs 6 hours after administration of 0.1 mg kg-1 Luc mRNA. (One-tailed Student's t-test, *=p<0.05, each dot represents a biologically independent replicate, with 4-7 biologically independent replicates per treatment). [Figure 6] Figure 1 shows that LNP-SNA mRNA expression profile is sequence dependent. (A) Luciferase (Luc) mRNA in liver, lung, and spleen by treatment. Luc mRNA was detected in harvested organs 6 hours after administration of B-19 formulation of LNP or LNP-SNA at 0.1 mg kg-1. (B) LNP and T-SNA show significant hepatic mRNA expression, but G-SNA does not. (C) G-SNA shows mRNA expression in spleen at levels comparable to LNP and T-SNA. (T-SNA is LNP functionalized with T21-SH DNA, G-SNA is LNP functionalized with (GGT)7-SH DNA sequence, N=4 biologically independent replicates). [Figure 7] 1 provides a depiction of the Cre system described in Example 2. [Figure 8] We show that LNP-SNAs functionalized with the outer (GGT)7 DNA sequence did not induce significant hepatic genome editing. This was shown by liver flow cytometry data from Ai14 mice 2 days after injection of 0.3 mg kg-1 Cre mRNA. (A) Measurement of tdTom positive cells in endothelial cells. (B) tdTom positive cells in hepatocytes. (C) tdTom positive cells in hepatic B cells. (D) tdTom positive cells in Kupffer cells. [Figure 9]Figure 9 shows that LNP-SNAs functionalized with the GGT sequence induced genome editing by Cre mRNA in splenic monocytes. Figure 9 displays spleen flow cytometry data from Ai14 mice 2 days after injection of 0.3 mg kg-1 Cre mRNA. [Figure 10] 13 presents the results of experiments demonstrating that the (GGT)7 outer sequence and DOPE helper lipid enabled enhanced delivery of LNP-SNAs to major splenic cell types. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] To treat disease, DNA and RNA therapeutics need to be delivered to target tissues and provide sustained benefits without side effects. Lipid nanoparticle spherical nucleic acids address this unmet need by using DNA and RNA sequences for nanoparticle targeting and tissue specificity. Lipid SNA structures have significantly different biodistribution properties than both lipid particles (loaded with nucleic acid) or traditional SNAs (liposomes and gold cores).

[0017] As used herein, a "targeting oligonucleotide" is an oligonucleotide that directs the LNP-SNA to a particular tissue and / or a particular cell type. In some embodiments, the targeting oligonucleotide is an aptamer. Thus, in some embodiments, the LNP-SNA of the present disclosure comprises an aptamer attached to the exterior of the lipid nanoparticle core, where the aptamer is designed to bind to one or more receptors on the surface of a particular cell type. In some embodiments, the targeting oligonucleotide is a nucleotide sequence similar to that of (GGT) n In a further embodiment, the targeting oligonucleotide comprises or consists of the nucleotide sequence, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, or is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. nIn a further embodiment, the targeting oligonucleotide comprises or consists of the nucleotide sequence: (GGT) n wherein n is 7. In some embodiments, the targeting oligonucleotide is a peptide oligonucleotide conjugate or an oligonucleotide small molecule conjugate.

[0018] As used herein, an "immunostimulatory oligonucleotide" is an oligonucleotide that can stimulate (e.g., induce or enhance) an immune response. Typical examples of immunostimulatory oligonucleotides are CpG motif-containing oligonucleotides, single-stranded RNA oligonucleotides, double-stranded RNA oligonucleotides, and double-stranded DNA oligonucleotides. A "CpG motif" is a cytosine-guanine dinucleotide sequence. Single-stranded RNA sequences can be recognized by toll-like receptors 8 and 9, double-stranded RNA sequences can be recognized by toll-like receptor 3, and double-stranded DNA can be recognized by toll-like receptor 3 and cyclic GMP-AMP synthase (cGAS).

[0019] The term "inhibitory oligonucleotide" refers to an oligonucleotide that reduces the production or expression of a protein, for example, by preventing the translation of mRNA into a protein in a ribosome, or that is sufficiently complementary to either the gene or mRNA encoding one or more of the target proteins to specifically bind (hybridize) to one or more target genes or mRNAs, thereby reducing the expression or biological activity of the target protein. Inhibitory oligonucleotides include, but are not limited to, isolated or synthetic short hairpin RNA (shRNA or DNA), antisense oligonucleotides (e.g., antisense RNA or DNA, chimeric antisense DNA or RNA), miRNA and miRNA mimics, small interfering RNA (siRNA), DNA or RNA inhibitors of innate immune receptors, aptamers, DNAzymes, or aptazymes.

[0020] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0021] The terms "polynucleotide" and "oligonucleotide" are interchangeable when used herein.

[0022] As used herein, the term "about," when used to modify a particular value or range, generally means within 20 percent, e.g., within 10 percent, within 5 percent, within 4 percent, within 3 percent, within 2 percent, or within 1 percent of the stated value or range.

[0023] Unless otherwise specified, all ranges contemplated herein include both endpoints and all numbers between the endpoints. The use of "about" or "approximately" in connection with a range applies to both ends of the range. Thus, "about 20 to 30" is intended to cover "about 20 to about 30," including at least the specified endpoints.

[0024] Lipid Nanoparticle Spherical Nucleic Acid (LNP-SNA) Lipid nanoparticle spherical nucleic acid (LNP-SNA) is composed of a lipid nanoparticle core decorated with oligonucleotides. The lipid nanoparticle core includes encapsulated oligonucleotides, ionizable lipids, phospholipids, sterols, and lipid-polyethylene glycol (lipid-PEG) conjugates. Due to the combination of core and shell properties, the construct has advantages over conventional liposomal SNA and gold SNA, for example, but not limited to, nucleic acid delivery. The oligonucleotides of the oligonucleotide shell can be oriented in various directions. In some embodiments, the oligonucleotides are oriented radially outward. The oligonucleotide shell includes one or more oligonucleotides bound to the outer surface of the lipid nanoparticle core. The spherical architecture of the oligonucleotide shell confers unique advantages over conventional nucleic acid delivery methods, including entry into almost all cells independent of transfection agents, resistance to nuclease degradation, sequence-based function, targeting, and diagnostics.

[0025] In some aspects, the present disclosure provides lipid nanoparticle spherical nucleic acid (LNP-SNA) comprising a lipid nanoparticle core and an oligonucleotide shell composed of oligonucleotides bound to the exterior of the lipid nanoparticle core, the lipid nanoparticle core comprising encapsulated oligonucleotides, ionizable lipids, phospholipids, sterols, and lipid-polyethylene glycol (lipid-PEG) conjugates, and at least 10% of the oligonucleotides in the oligonucleotide shell are covalently bound to the exterior of the lipid nanoparticle core via the lipid-PEG conjugates. In various embodiments, the LNP-SNA comprises a targeting peptide, a targeting antibody, or a combination thereof bound to the exterior of the lipid nanoparticle core. Such targeting peptides are, for example, but not limited to, antibody Fab fragments that bind to surface markers of target cells and peptides designed to be charged in different pH environments. In various embodiments, one or more gene editing oligonucleotides are encapsulated in the lipid nanoparticle core of the LNP-SNA. Such gene editing oligonucleotides are, for example, but not limited to, messenger RNA (mRNA) encoding a gene editor protein, a DNA or RNA gene editor substrate (e.g., guide RNA), or a combination thereof.

[0026] Thus, the lipid nanoparticle core comprises an encapsulated oligonucleotide, an ionizable lipid, a phospholipid, a sterol, and a lipid-polyethylene glycol (lipid-PEG) conjugate. In some embodiments, the ionizable lipid is dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), C12-200, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), similar lipid / lipidoid structures, or combinations thereof. In some embodiments, the ionizable lipid is dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA). In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dihexadecanoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or a combination thereof. In any of the aspects or embodiments of the present disclosure, the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).In a further embodiment, the sterol is 3β-hydroxycholest-5-ene (cholesterol), 9,10-secocholesta-5,7,10(19)-trien-3β-ol (vitamin D3), 9,10-secoegosta-5,7,10(19),22-tetraen-3β-ol (vitamin D2), calcipotriol, 24-ethyl-5,22-cholestadien-3β-ol (stigmasterol), 22,23-dihydrostigmasterol (β-sitosterol), 3,28-dihydroxy-lupeol (beta-sitosterol), 5,5-dihydroxy-1,1,2,3,4,4-trimethylol, 5,5-dihydro ... Turin), lupeol, ursolic acid, oleanolic acid, 24α-methylcholesterol (campesterol), 24-ethylcholesta-5,24(28)E-dien-3β-ol (fucosterol), 24-methylcholesta-5,22-dien-3β-ol (brassicasterol), 24-methylcholesta-5,7,22-trien-3β-ol (ergosterol), 9,11-dehydroergosterol, daucosterol, or any of the foregoing sterols modified with one or more amino acids. In some embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate comprises a 2000 Dalton (Da) polyethylene glycol. In further embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate is a lipid-PEG-maleimide. In still further embodiments, the lipid-PEG-maleimide is 1,2-dipalmitryl-sn-glycero-3-phosphoethanolamine (DPPE) conjugated to a 2000 Da polyethylene glycol maleimide, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) conjugated to a 2000 Da polyethylene glycol maleimide, or a combination thereof.

[0027] LNP-SNAs may range from about 1 nanometer (nm) to about 500 nm, about 1 nm to about 400 nm, about 1 nm to about 300 nm, about 1 nm to about 200 nm, about 1 nm to about 150 nm, about 1 nm to about 100 nm, about 1 nm to about 90 nm, about 1 nm to about 80 nm in diameter, about 1 nm to about 70 nm in diameter, about 1 nm to about 60 nm in diameter, about 1 nm to about 50 nm in diameter, about 1 nm to about 40 nm in diameter, about 1 nm to about 30 nm in diameter, about 1 nm to about 20 nm in diameter, about 1 nm to about 10 nm, about 10 nm to about 150 The diameter of the nanoparticles may be in the range of about 10 nm to about 140 nm, about 10 nm to about 130 nm, about 10 nm to about 120 nm, about 10 nm to about 110 nm, about 10 nm to about 100 nm, about 10 nm to about 90 nm, about 10 nm to about 80 nm, about 10 nm to about 70 nm, about 10 nm to about 60 nm, about 10 nm to about 50 nm, about 10 nm to about 40 nm, about 10 nm to about 30 nm, or about 10 nm to about 20 nm. In some embodiments, the LNP-SNA is 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, or 10 nm in diameter, or is at least 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, or 10 nm in diameter, or is less than about 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, or 10 nm in diameter (or is the average diameter if multiple LNP-SNAs are present).In some embodiments, the size of the LNP-SNAs ranges from about 10 nm to about 150 nm (average diameter), from about 10 nm to about 140 nm, from about 10 nm to about 130 nm, from about 10 nm to about 120 nm, from about 10 nm to about 110 nm, from about 10 nm to about 100 nm, from about 10 nm to about 90 nm, from about 10 nm to about 80 nm, from about 10 nm to about 70 nm, from about 10 nm to about 60 nm, from about 10 nm to about 50 nm, or the like. The average diameter of the LNP-SNA may be about 10 nm to about 40 nm, about 10 nm to about 30 nm, about 10 nm to about 20 nm, about 40 nm to about 150 nm, about 40 nm to about 100 nm, about 40 nm to about 90 nm, about 40 nm to about 80 nm, about 50 nm to about 200 nm, about 50 nm to about 150 nm, about 50 nm to about 100 nm, about 50 nm to about 90 nm, or about 50 nm to about 80 nm. In some embodiments, the diameter of the LNP-SNA (or the average diameter of a plurality of LNP-SNAs) is about 40 nm to about 150 nm, about 50 to about 200 nm, or about 40 to about 100 nm. In some embodiments, the size of the nanoparticles used in the methods may vary as needed depending on their particular use or application. Variation in size is advantageously used to optimize certain physical properties of the LNP-SNA, such as the amount of surface area to which oligonucleotides can be attached, as described herein. It will be understood that the aforementioned diameters of the LNP-SNA can apply to the diameter of the lipid nanoparticle core itself, or the diameter of the lipid nanoparticle core and a shell of oligonucleotides attached thereto.

[0028] Oligonucleotides The present disclosure provides a lipid nanoparticle spherical nucleic acid (LNP-SNA) comprising a lipid nanoparticle core and a shell of oligonucleotides attached to the exterior of the lipid nanoparticle core. As described herein, the lipid nanoparticle core comprises an encapsulated oligonucleotide. In any of the aspects or embodiments of the present disclosure, the oligonucleotide is selected from the group consisting of (GGT) nIn a further embodiment, the targeting oligonucleotide comprises or consists of the nucleotide sequence, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, or is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. n In a further embodiment, the oligonucleotide comprises or consists of the nucleotide sequence: (GGT) n In various embodiments, the nucleotide sequence comprises or consists of the nucleotide sequence: (GGT) n The sequence is at the 5' or 3' end of the oligonucleotide. In various embodiments, (GGT) n The sequence is proximal or distal to the nanoparticle core. In various embodiments, (GGT) n The sequence is at the end of the oligonucleotide attached to the nanoparticle core, (GGT) n The sequence is at the end of the oligonucleotide that is not attached to the nanoparticle core, or both. In various embodiments, the shell of the oligonucleotide comprises an inhibitory oligonucleotide, an immunostimulatory oligonucleotide, a targeting oligonucleotide, or a combination thereof. In various embodiments, the oligonucleotide of the present disclosure comprises a DNA oligonucleotide, an RNA oligonucleotide, a modified form thereof, or a combination thereof. In any aspect or embodiment described herein, the oligonucleotide is single-stranded, double-stranded, or partially double-stranded. In any aspect or embodiment of the present disclosure, the oligonucleotide comprises a detectable marker.

[0029] Modified forms of oligonucleotides, including those with at least one modified internucleotidic linkage as described herein, are also contemplated by the present disclosure. In some embodiments, the oligonucleotide is all or partly a peptide nucleic acid. Other modified internucleoside linkages include at least one phosphorothioate linkage. Still other modified oligonucleotides include those that include one or more universal bases. A "universal base" refers to a molecule that can replace a linkage to any one of A, C, G, T, and U in a nucleic acid by forming a hydrogen bond without significant structural destabilization. Oligonucleotides incorporating universal base analogs can function, for example, as probes in hybridization. Examples of universal bases include, but are not limited to, 5'-nitroindole-2'-deoxyriboside, 3-nitropyrrole, inosine, and hypoxanthine.

[0030] The term "nucleotide" or its plural forms as used herein are interchangeable with the variants discussed herein and otherwise known in the art. The term "nucleobase" or its plural forms as used herein are interchangeable with the variants discussed herein and otherwise known in the art. Nucleotides or nucleobases include the naturally occurring nucleobases A, G, C, T, and U. Non-naturally occurring nucleobases include, for example, but are not limited to, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosine, N',N'-ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C3-C6)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, and the "non-naturally occurring" nucleobases described in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freier and Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp 4429-4443. The term "nucleobase" includes the known purine and pyrimidine heterocycles, as well as their heterocyclic analogs and tautomers.Additionally, naturally occurring and non-naturally occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan et al.), Chapter 15 of Sanghvi, Antisense Research and Application, Ed. S.T. Crooke and B. Lebleu, CRC Press, 1993, Englisch et al., 1991, Angewandte Chemie, International Edition, 30:613-722 (see especially pages 622 and 623), and the Concise Encyclopedia of Polymer Science and Engineering, J.I. Kroschwitz Ed., John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design 1991, 6, 585-607, each of which is incorporated herein by reference in its entirety. In various embodiments, oligonucleotides also contain one or more "nucleoside bases" or "base units", which are a category of non-naturally occurring nucleotides that contain compounds such as heterocyclic compounds that are not nucleoside bases in the most classical sense, but can function as nucleoside bases, including certain "universal bases".Universal bases include 3-nitropyrrole, optionally substituted indoles (e.g., 5-nitroindoles), and optionally substituted hypoxanthines.Other desirable universal bases include pyrrole, diazole, or triazole derivatives that contain universal bases known in the art.

[0031] Examples of oligonucleotides include those containing modified backbones or non-natural internucleoside linkages. Oligonucleotides with modified backbones include those that have a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are considered to be within the meaning of "oligonucleotide."

[0032] Modified oligonucleotide backbones containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phototriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs thereof, and those having reverse polarity where one or more internucleotide linkages are 3'-3', 5'-5' or 2'-2' linkages. Also contemplated are oligonucleotides having only one inverted nucleoside residue that contains a single 3'-3' linkage at the 3'-most internucleotide linkage, i.e., that may be abbasic (either no nucleotide or a hydroxyl group in that location). Salts, mixed salts, and free acid forms are also contemplated. Representative United States patents which teach the preparation of the above phosphorus-containing linkages include U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, Nos. 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361, 5,194,599, 5,565,555, 5,527,899, 5,721,218, 5,672,697 and 5,625,050, the disclosures of which are incorporated herein by reference.

[0033] Modified oligonucleotide backbones that do not contain phosphorus atoms therein have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include backbones with morpholino linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and methylenethioformacetyl backbones; riboacetyl backbones; alkene-containing backbones; sulfamic acid backbones; methyleneimino and methylenehydrazino backbones; sulfonic acid and sulfonamide backbones; amide backbones; and N, O, S and CH 2 Other scaffolds exist that have a mixture of component parts, e.g., U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, 5,59 See Nos. 6,086, 5,602,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, 5,792,608, 5,646,269 and 5,677,439, the disclosures of which are incorporated herein by reference.

[0034] In yet further embodiments, there are oligonucleotide mimetics in which one or more sugars and / or one or more internucleotide bonds of nucleotide units are both replaced with "non-naturally occurring" groups. The bases of the oligonucleotide are maintained for hybridization. In some aspects, this embodiment contemplates peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced with an amide-containing backbone. See, for example, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, and Nielsen et al., Science, 1991, 254, 1497-1500, the disclosures of which are incorporated herein by reference.

[0035] In still further embodiments, oligonucleotides having phosphorothioate backbones and oligonucleosides having heteroatom backbones are provided, including those having the -CH 2 -NH-O-CH 2 -, -CH 2 -N(CH 3 )-O-CH 2 -, -CH 2 -ON(CH 3 )-CH 2 -, -CH 2 -N(CH 3 )-N(CH 3 )-CH 2 - and -ON(CH 3 )-CH 2 -CH 2 Also contemplated are oligonucleotides having morpholino backbone structures, as described in U.S. Patent No. 5,034,506.

[0036] In various forms, the bond between two consecutive monomers in an oligonucleotide is -CH 2 -, -O-, -S-, -NR H -, >C=O, >C=NR H , >C=S, -Si(R”) 2 -, -SO-, -S(O) 2 -, -P(O)2 -, -PO(BH 3 )-, -P(O,S)-, -P(S) 2 -, -PO(R")-, -PO(OCH 3 )- and -PO(NHR H ) , wherein R H is hydrogen and C 1~4 -alkyl, R″ is selected from C 1~6 -alkyl and phenyl. An illustrative example of such a bond is -CH 2 -CH 2 -CH 2 -, -CH 2 -CO-CH 2 -, -CH 2 -CHOH-CH 2 -, -O-CH 2 -O-, -O-CH 2 -CH 2 -, -O-CH 2 -CH=(R when used as a bond to a subsequent monomer 5 ), -CH 2 -CH 2 -O-, -NR H -CH 2 -CH 2 -, -CH 2 -CH 2 -NR H -, -CH 2 -NR H -CH 2 --, -O-CH 2 -CH 2 -NR H -, -NR H -CO-O-, -NR H -CO-NR H -, -NR H -CS-NR H -, -NR H -C(=NR H )-NR H -, -NR H -CO-CH 2 -NR H -O-CO-O-, -O-CO-CH 2 -O-, -O-CH 2 -CO-O-, -CH2 -CO-NR H -, -O-CO-NR H -, -NR H -CO-CH 2 -, -O-CH 2 -CO-NR H -, -O-CH 2 -CH 2 -NR H -, -CH=NO-, -CH 2 -NR H -O-, -CH 2 -ON=(R when used as a bond to a subsequent monomer) 5 ), -CH 2 -O-NR H -, -CO-NR H -CH 2 -, -CH 2 -NR H -O-, -CH 2 -NR H -CO-, -O-NR H -CH 2 -, -O-NR H , -O-CH 2 -S-, -S-CH 2 -O-, -CH 2 -CH 2 -S-, -O-CH 2 -CH 2 -S-, -S-CH 2 -CH=(R when used as a bond to a subsequent monomer 5 ), -S-CH 2 -CH 2 -,-S-CH 2 -CH 2 --O-, -S-CH 2 -CH 2 -S-, -CH 2 -S-CH 2 -, -CH 2 -SO-CH 2 -, -CH 2 -SO 2 -CH 2 -, -O-SO-O-, -OS(O) 2 -O-, -OS(O) 2 -CH 2 -, -OS(O) 2-NR H -,-NR H -S(O) 2 -CH 2 -;-O-S(O) 2 -CH 2 -,-O-P(O) 2 -O-,-O-P(O,S)-O-,-O-P(S) 2 -O-,-S-P(O) 2 -O-,-S-P(O,S)-O-,-S-P(S) 2 -O-,-O-P(O) 2 -S-,-O-P(O,S)-S-,-O-P(S) 2 -S-,-S-P(O) 2 -S-,-S-P(O,S)-S-,-S-P(S) 2 -S-,-O-PO(R”)-O-,-O-PO(OCH 3 )-O-,-O-PO(O CH 2 CH 3 )-O-,-O-PO(O CH 2 CH 2 S-R)-O-,-O-PO(BH 3 )-O-,-O-PO(NHR N )-O-,-O-P(O) 2 -NR H H-,-NR H -P(O) 2 -O-,-O-P(O,NR H )-O-,-CH 2 -P(O) 2 -O-,-O-P(O) 2 -CH 2 -、and -O-Si(R”) 2 -O- and, among these, -CH 2 -CO-NR H -,-CH 2 -NR H -O-,-S-CH 2 -O-,-O-P(O) 2 -O-O-P(-O,S)-O-,-O-P(S) 2 -O-,-NR H P(O) 2 -O-,-O-P(O,NR H )-O-,-O-PO(R”)-O-,-O-PO(CH3 )-O- and -O-PO(NHR N )-O-(wherein, R H is hydrogen and C 1~4 -alkyl, R″ is selected from C 1~6 -alkyl and phenyl) are contemplated. Further examples are provided in Mesmaeker et.al., Current Opinion in Structural Biology 1995, 5, 343-355 and Susan M. Freier and Karl-Heinz Altmann, Nucleic Acids Research, 1997, vol 25, pp 4429-4443.

[0037] Still other modified forms of oligonucleotides are described in detail in US Patent Application Publication No. 2004 / 0219565, the disclosure of which is incorporated herein by reference in its entirety.

[0038] Modified oligonucleotides may also contain one or more substituted sugar moieties. In certain embodiments, the oligonucleotides contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C 1 ~C 10 Alkyl or C 2 ~C 10 It can be alkenyl and alkynyl. In other embodiments, O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH2 ) n ON[(CH 2 ) n CH 3 ] 2 where n and m are from 1 to about 10. Other oligonucleotides include one of the following at the 2' position: 1 ~C 10 Lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , S.O. 2 CH 3 , O.N.O. 2 , NO 2 , N 3 , NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, or an RNA cleaving group. In one aspect, the modification includes 2'-methoxyethoxy (also known as 2'-O-(2-methoxyethyl) or 2'-MOE, 2'-O-CH 2 CH 2 OCH 3 ) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., alkoxyalkoxy groups. Other modifications include O(CH), also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE. 2 ) 2 ON(CH 3 ) 2 groups, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH 2 -O-CH 2 -N(CH 3 ) 2 Examples include:

[0039] Further modifications include 2'-methoxy (2'-O-CH 3 ), 2'-aminopropoxy (2'-OCH2 CH 2 CH 2 NH 2 ), 2'-allyl (2'-CH 2 -CH=CH 2 ), 2'-O-allyl (2'-O-CH 2 -CH=CH 2 ), and 2'-fluoro (2'-F). The 2' modification can be at the arabino (up) or ribo (down) position. In one aspect, the 2'-arabino modification is a 2'-F. Similar modifications can also be made at other positions on oligonucleotides, such as the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and at the 5' position of the 5' terminal nucleotide. Oligonucleotides can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. See, for example, U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,576,427, 5,591,7 22, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, 5,792,747, and 5,700,920, the disclosures of which are incorporated herein by reference in their entireties.

[0040] In some embodiments, sugar modifications include Locked Nucleic Acids (LNAs), in which a 2'-hydroxyl group is attached to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. In certain embodiments, the linkage is a methylene (-CH 2 -) n where n is 1 or 2. LNAs and their preparation are described in WO98 / 39352 and WO99 / 14226.

[0041] Modified nucleotides are described in EP 1072679 and WO 97 / 12896, the disclosures of which are incorporated herein by reference. Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (succinimide), ... douracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases can also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, Englisch et al., 1991, Angewandte Chemie, International Edition, 30:613, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993. Some of these bases are useful for increasing binding affinity and include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C, and in certain embodiments are combined with 2'-O-methoxyethyl sugar modifications. See U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, and 5,55 See, for example, Nos. 2,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,830,653, 5,763,588, 6,005,096, 5,750,692, and 5,681,941, the disclosures of which are incorporated herein by reference.

[0042] Methods for making polynucleotides of a given sequence are well known. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press, New York, 1991). Solid-phase synthesis methods are suitable for both polyribonucleotides and polydeoxyribonucleotides (the well-known methods for DNA synthesis are also useful for RNA synthesis). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can also be incorporated into polynucleotides. See, e.g., U.S. Pat. No. 7,223,833; Katz, J. Am. Chem. Soc., 74:2238 (1951); Yamane, et al., J. Am. Chem. Soc., 83:2599 (1961); Kosturko, et al., Biochemistry, 13:3949 (1974); Thomas, J. Am. Chem. Soc., 76:6032 (1954); Zhang, et al., J. Am. Chem. Soc., 127:74-75 (2005); and Zimmermann, et al., J. Am. Chem. Soc., 124:13684-13685 (2002).

[0043] In various embodiments, the oligonucleotides of the present disclosure (e.g., oligonucleotides in an oligonucleotide shell or encapsulated oligonucleotides), or modified forms thereof, are generally from about 10 to about 100 nucleotides in length. More specifically, the oligonucleotides of the present disclosure may be from about 5 to about 90 nucleotides in length, from about 5 to about 80 nucleotides in length, from about 5 to about 70 nucleotides in length, from about 5 to about 60 nucleotides in length, from about 5 to about 50 nucleotides in length, from about 5 to about 45 nucleotides in length, from about 5 to about 40 nucleotides in length, from about 5 to about 35 nucleotides in length, from about 5 to about 30 nucleotides in length, from about 5 to about 25 nucleotides in length, from about 5 to about 20 nucleotides in length, from about 5 to about 15 nucleotides in length, from about 5 to about 10 nucleotides in length, from about 10 to about 90 nucleotides in length, from about 10 to about 8 ... The oligonucleotides may be 0 to about 70 nucleotides in length, about 10 to about 60 nucleotides in length, about 10 to about 50 nucleotides in length, about 10 to about 45 nucleotides in length, about 10 to about 40 nucleotides in length, about 10 to about 35 nucleotides in length, about 10 to about 30 nucleotides in length, about 10 to about 25 nucleotides in length, about 10 to about 20 nucleotides in length, about 10 to about 15 nucleotides in length, about 18 to about 28 nucleotides in length, about 15 to about 26 nucleotides in length, and all oligonucleotide intermediate lengths of the specifically disclosed sizes to the extent that the oligonucleotide achieves the desired result. In further embodiments, the oligonucleotides of the present disclosure (e.g., encapsulated oligonucleotides) are from about 5 nucleotides to about 5000 nucleotides in length.In further embodiments, the oligonucleotides of the present disclosure are from about 5 to about 4000 nucleotides in length, from about 5 to about 3500 nucleotides in length, from about 5 to about 3000 nucleotides in length, from about 5 to about 2500 nucleotides in length, from about 5 to about 2000 nucleotides in length, from about 5 to about 1500 nucleotides in length, from about 5 to about 1000 nucleotides in length, from about 5 to about 900 nucleotides in length, from about 5 to about 800 nucleotides in length, from about 5 to about 700 nucleotides in length, from about 5 to about 600 nucleotides in length, from about 5 to about 500 nucleotides in length, from about 5 to about 450 nucleotides in length, from about 5 to about 400 nucleotides in length, from about 5 to about 350 nucleotides in length, from about 5 to about 50 ... about 5 to about 300 nucleotides in length, about 5 to about 250 nucleotides in length, about 5 to about 200 nucleotides in length, about 5 to about 150 nucleotides in length, about 5 to about 100 nucleotides in length, about 5 to about 90 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 70 nucleotides in length, about 5 to about 60 nucleotides in length, about 5 to about 50 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 10 nucleotides in length, and all oligonucleotide intermediate lengths of the specifically disclosed sizes to the extent that the oligonucleotide achieves the desired result. Thus, in various embodiments, the oligonucleotides of the present disclosure comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 , 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1700, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more nucleotides in length, or at least those nucleotides in length.In further embodiments, the oligonucleotides of the disclosure are 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 1 The nucleic acid sequence is less than 6, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more nucleotides in length. In various embodiments, the shell of oligonucleotides bound to the exterior of the lipid nanoparticle core of the LNP-SNA comprises a plurality of oligonucleotides all having the same length / sequence, and in some embodiments, the plurality of oligonucleotides comprises one or more oligonucleotides having a different length and / or sequence relative to at least one other oligonucleotide in the plurality. In various embodiments, the lipid nanoparticle core comprises a plurality of oligonucleotides encapsulated therein all having the same length / sequence, while in some embodiments, the lipid nanoparticle core comprises one or more oligonucleotides having a different length and / or sequence relative to at least one other oligonucleotide in the plurality. For example, but not limited to, in some embodiments, the lipid nanoparticle core comprises an mRNA encoding a gene editing endonuclease (e.g., cas9) in combination with a substrate guide RNA encapsulated therein for gene editing.

[0044] In some embodiments, the oligonucleotide (e.g., oligonucleotides in the shell of an oligonucleotide and / or encapsulated in a lipid nanoparticle) is an aptamer. Thus, all the features and aspects of oligonucleotides described herein (e.g., length, type (DNA, RNA, modified forms thereof), optional presence of spacers) also apply to aptamers. Aptamers are oligonucleotide sequences that can be evolved to bind to various target analytes of interest. Aptamers may be single-stranded, double-stranded, or partially double-stranded.

[0045] Methods for binding the detectable markers (eg, fluorophores, radiolabels) and therapeutic agents (eg, antibodies) described herein to oligonucleotides are known in the art.

[0046] In various aspects, the LNP-SNA of the present disclosure has the ability to bind to multiple targets (e.g., polynucleotides, proteins). In some embodiments, the LNP-SNA further comprises one or more oligonucleotides that are inhibitory oligonucleotides as described herein. Such inhibitory oligonucleotides, in various embodiments, are present within a shell of oligonucleotides bound to the exterior of the lipid nanoparticle core, encapsulated in the lipid nanoparticle core, or both. Thus, in some embodiments, the LNP-SNA of the present disclosure comprises one or more oligonucleotides that have a sequence sufficiently complementary to a target polynucleotide to hybridize under the conditions used. In some embodiments, the LNP-SNA comprises two or more oligonucleotides that are not identical, i.e., at least one of the oligonucleotides differs from at least one other oligonucleotide in that it has a different length and / or a different sequence. For example, when a specific polynucleotide is targeted, a single LNP-SNA has the ability to bind to multiple copies of the same target. In some embodiments, a single LNP-SNA has the ability to bind to different targets. Thus, in various aspects, a single LNP-SNA may be used in a method to inhibit the expression of two or more gene products. In various embodiments, oligonucleotides are therefore used to target particular polynucleotides, whether at one or more specific regions of the target polynucleotide, or, optionally, across the entire length of the target polynucleotide, to effect a desired level of inhibition of gene expression.

[0047] In some embodiments, the LNP-SNA further comprises one or more oligonucleotides that are immunostimulatory oligonucleotides as described herein. Such immunostimulatory oligonucleotides are in various embodiments present within a shell of oligonucleotides bound to the exterior of the lipid nanoparticle core, encapsulated in the lipid nanoparticle core, or both. Thus, in various aspects and embodiments of the present disclosure, the LNP-SNAs of the present disclosure have immunostimulatory activity, inhibition of gene expression activity, or both. The immunostimulatory oligonucleotides are, in any of the aspects or embodiments of the present disclosure, CpG motif-containing oligonucleotides. In various embodiments, the CpG motif-containing oligonucleotides are class A CpG oligonucleotides, class B CpG oligonucleotides, or class C CpG oligonucleotides. In various embodiments, the LNP-SNAs comprise inhibitory oligonucleotides, immunostimulatory oligonucleotides, targeting oligonucleotides, or mixtures thereof.

[0048] Lipid Nanoparticle Surface / Encapsulation Density. A surface density sufficient to stabilize the nanoparticles and the conditions necessary to obtain it for a desired combination of nanoparticles and oligonucleotides can be empirically determined. Generally, the oligonucleotides of the present disclosure have a surface density of at least about 2 picomoles / cm. 2 In some embodiments, the surface density is about 15 picomoles / cm. 2 or at least about 15 picomoles / cm 2 The oligonucleotide is at least 2 pmol / cm 2 , at least 3 pmol / cm 2 , at least 4 pmol / cm 2 , at least 5 pmol / cm 2 , at least 6 pmol / cm 2 , at least 7 pmol / cm 2 , at least 8 pmol / cm 2 , at least 9 pmol / cm 2 , at least 10 pmol / cm 2 , at least about 15 pmol / cm2 , at least about 19 pmol / cm 2 , at least about 20 pmol / cm 2 , at least about 25 pmol / cm 2 , at least about 30 pmol / cm 2 , at least about 35 pmol / cm 2 , at least about 40 pmol / cm 2 , at least about 45 pmol / cm 2 , at least about 50 pmol / cm 2 , at least about 55 pmol / cm 2 , at least about 60 pmol / cm 2 , at least about 65 pmol / cm 2 , at least about 70 pmol / cm 2 , at least about 75 pmol / cm 2 , at least about 80 pmol / cm 2 , at least about 85 pmol / cm 2 , at least about 90 pmol / cm 2 , at least about 95 pmol / cm 2 , at least about 100 pmol / cm 2 , at least about 125 pmol / cm 2 , at least about 150 pmol / cm 2 , at least about 175 pmol / cm 2 , at least about 200 pmol / cm 2 , at least about 250 pmol / cm 2 , at least about 300 pmol / cm 2 , at least about 350 pmol / cm 2 , at least about 400 pmol / cm 2 , at least about 450 pmol / cm 2 , at least about 500 pmol / cm 2 , at least about 550 pmol / cm 2 , at least about 600 pmol / cm 2 , at least about 650 pmol / cm 2 , at least about 700 pmol / cm 2 , at least about 750 pmol / cm 2 , at least about 800 pmol / cm2 , at least about 850 pmol / cm 2 , at least about 900 pmol / cm 2 , at least about 950 pmol / cm 2 , at least about 1000 pmol / cm 2Also provided is a method for binding the oligonucleotides to the exterior of the lipid nanoparticle core at a surface density of at least 1000. Alternatively, the density of the oligonucleotides bound to the exterior of the lipid nanoparticle core is measured by the number of oligonucleotides bound to the LNP-SNA. With respect to the surface density of the oligonucleotides bound to the LNP-SNA, it is contemplated that the LNP-SNA described herein comprises about 1 to about 2,500, or about 1 to about 1,000, or about 1 to about 500 oligonucleotides bound to the exterior of the lipid nanoparticle core. In various embodiments, the LNP-SNA comprises about 10 to about 500, or about 10 to about 450, or about 10 to about 400, or about 10 to about 300, or about 10 to about 200, or about 10 to about 190, or about 10 to about 180, or about 10 to about 170, or about 10 to about 160, or about 10 to about 150, or about 10 to about 140, or about 10 to about 130, or about 10 to about 120, or about 10 to about 110, or about 10 to about 100, or about 10 to about 90, or about 10 to about 80, or about 10 to about 70, or about 10 to about 60, or about 10 to about 50, or about 10 to about 40, or about 10 to about 30, or about 10 to about 20 oligonucleotides. In some embodiments, the LNP-SNA comprises about 80 to about 500, or about 80 to about 400 oligonucleotides within a shell of oligonucleotides bound to the exterior of the lipid nanoparticle core. In further embodiments, the LNP-SNAs comprise at least about 5, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 oligonucleotides within a shell of oligonucleotides attached to the exterior of the lipid nanoparticle core.In further embodiments, the LNP-SNAs comprise or consist of at least about 1, 2, 3, 4, 5, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 127, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 oligonucleotides within a shell of oligonucleotides attached to the exterior of the lipid nanoparticle core. In still further embodiments, the shell of oligonucleotides bound to the lipid nanoparticle core of the LNP-SNA comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 200, 300, 400, 500, or more oligonucleotides, in some embodiments, the shell of oligonucleotides bound to the lipid nanoparticle core of the LNP-SNA comprises about 400 oligonucleotides. In some embodiments, the shell of oligonucleotides attached to the lipid nanoparticle core of the LNP-SNA comprises or consists of about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or more oligonucleotides. In various embodiments, about 2 to about 1000, or about 2 to about 500, or about 100 to about 1000, or about 50 to about 1000, or about 100 to about 500 oligonucleotides are attached to the outer surface of the lipid nanoparticle core.In further embodiments, the number of nuclei is from about 10 to about 1000, or from about 10 to about 750, or from about 10 to about 500, or from about 10 to about 400, or from about 10 to about 250, or from about 10 to about 100, or from about 50 to about 1000, or from about 50 to about 750, or from about 50 to about 500, or from about 50 to about 250, or from about 100 to about 1000, or from about 100 to about 500, or from about 2 to about 90, or from about 2 to about 80, or from about 2 to about 70, or from about 2 to about 60, or from about 2 to about 50, or from about 2 to about 40, or from about 2 to about 30, or from about 2 to about 2 0, or about 2 to about 10, or about 10 to about 100, or about 10 to about 90, or about 10 to about 80, or about 10 to about 70, or about 10 to about 60, or about 10 to about 50, or about 10 to about 40, or about 10 to about 30, or about 10 to about 20, or about 20 to about 100, or about 20 to about 90, or about 20 to about 80, or about 20 to about 70, or about 20 to about 60, or about 20 to about 50, or about 20 to about 40, or about 20 to about 30 oligonucleotides are bound to the outer surface of the lipid nanoparticle core. In still further embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 oligonucleotides are attached to the exterior of the lipid nanoparticle core. With respect to the density of oligonucleotides encapsulated in the lipid nanoparticle core, it is contemplated that the LNP-SNAs described herein comprise about 1 to about 250, about 1 to about 220, about 1 to about 200, about 1 to about 150, about 1 to about 120, about 1 to about 100, about 1 to about 90, about 1 to about 80, about 1 to about 70, about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5 oligonucleotides encapsulated in the lipid nanoparticle core. In further embodiments, the LNP-SNAs of the present disclosure comprise about 10 to about 250, about 10 to about 220, about 10 to about 200, about 10 to about 150, about 10 to about 120, about 10 to about 100, about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 10 to about 30, or about 10 to about 20 oligonucleotides encapsulated in the lipid nanoparticle core.In further embodiments, the LNP-SNAs of the present disclosure comprise or consist of about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 120, 150, 170, 200, 220, 250 or more oligonucleotides encapsulated in the lipid nanoparticle core. In further embodiments, the LNP-SNAs of the present disclosure comprise less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 120, 150, 170, 200, 220, or 250 oligonucleotides encapsulated in the lipid nanoparticle core.

[0049] Spacer. In some aspects, oligonucleotides are attached to lipid nanoparticle cores via a spacer (and in some embodiments, via a linker). As used herein, "spacer" refers to a moiety that serves to increase the distance between nanoparticles and oligonucleotides, or to increase the distance between individual oligonucleotides when multiple copies are attached to nanoparticles. Thus, it is contemplated that spacers are located between individual oligonucleotides in tandem, regardless of whether the oligonucleotides have the same sequence or different sequences.

[0050] In some aspects, the spacer, when present, is an organic moiety. In some aspects, the spacer is a polymer, including but not limited to a water-soluble polymer, a nucleic acid, a polypeptide, an oligosaccharide, a carbohydrate, a lipid, ethyl glycol, or a combination thereof. In any of the aspects or embodiments of the present disclosure, the spacer is an oligo(ethylene glycol)-based spacer. In various embodiments, the oligonucleotide comprises one, two, three, four, five, or more spacer (e.g., spacer-18 (hexaethylene glycol)) moieties. In further embodiments, the spacer is an alkane-based spacer (e.g., C12). In some embodiments, the spacer is an oligonucleotide spacer (e.g., T5). The oligonucleotide spacer can have any sequence that does not interfere with the ability of the oligonucleotide to perform its intended function (e.g., stimulate an immune response or inhibit gene expression). In certain aspects, the bases of the oligonucleotide spacer are all adenylic acids, all thymidylic acids, all cytidylic acids, all guanylic acids, all uridylic acids, or all some other modified base.

[0051] In various embodiments, the length of the spacer is or is equal to at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, 5-10 nucleotides, 10 nucleotides, 20 nucleotides, 10-30 nucleotides, or even more than 30 nucleotides.

[0052] Conjugation of oligonucleotides to lipid nanoparticle cores. Oligonucleotides contemplated for use according to the present disclosure include those conjugated to the nanoparticle cores through any means (e.g., covalent or non-covalent). In any of the aspects or embodiments of the present disclosure, the oligonucleotides are conjugated to the exterior of the lipid nanoparticle cores via covalent attachment to lipid-polyethylene glycol (lipid-PEG) conjugates of the oligonucleotides. In some embodiments, 10% or at least 10% of the oligonucleotides in the oligonucleotide shell are covalently attached to the exterior of the lipid nanoparticle cores via lipid-PEG conjugates. In further embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the oligonucleotides in the oligonucleotide shell are covalently attached to the exterior of the lipid nanoparticle cores via lipid-PEG conjugates. In some embodiments, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the oligonucleotides in the oligonucleotide shell are covalently attached to the exterior of the lipid nanoparticle cores via lipid-PEG conjugates. In still further embodiments, less than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the oligonucleotides in the oligonucleotide shell are covalently attached to the exterior of the lipid nanoparticle core via lipid PEG-conjugates. In some embodiments, one or more oligonucleotides in the oligonucleotide shell are attached to the exterior of the lipid nanoparticle core via lipid anchor groups. The lipid anchor groups are attached to the 5' or 3' ends of the oligonucleotides in various embodiments. In various embodiments, the lipid anchor groups are cholesterol or tocopherol.

[0053] Regardless of the means by which the oligonucleotides are attached to the lipid nanoparticle core, attachment in various aspects is through a 5' linkage, a 3' linkage, some type of internal linkage, or any combination of these linkages. In some embodiments, the oligonucleotides are covalently attached to the nanoparticles. In further embodiments, the oligonucleotides are non-covalently attached to the nanoparticles. In further embodiments, the oligonucleotides are attached to the nanoparticles via a combination of covalent and non-covalent bonds.

[0054] Methods of binding are known to those skilled in the art and are described in US Publication No. 2009 / 0209629, the entirety of which is incorporated herein by reference. Methods of binding RNA to nanoparticles are generally described in PCT / US2009 / 65822, the entirety of which is incorporated herein by reference. Methods of associating oligonucleotides with liposome particles are described in US Patent Application Publication No. 2016 / 0310425, the entirety of which is incorporated herein by reference.

[0055] Synthesis of LNP-SNAs As described herein, the LNP-SNA of the present disclosure generally comprises a lipid nanoparticle core containing encapsulated oligonucleotides and a shell of oligonucleotides bound to the exterior of the lipid nanoparticle core, and at least 10% of the oligonucleotides in the shell of oligonucleotides are covalently bound to the exterior of the lipid nanoparticle core via lipid-PEG conjugates.Thus, the LNP-SNA of the present disclosure is synthesized such that the oligonucleotides are encapsulated in the lipid nanoparticle core and the shell of oligonucleotides is bound to the exterior of the lipid nanoparticle core.The synthesis of LNP-SNA is described in detail herein (e.g., Example 1) and is generally shown in Figure 1.

[0056] Generally, and by way of example, lipid nanoparticles (LNPs) may be formulated by diluting lipids and sterols in ethanol. The nucleic acid to be encapsulated is dissolved separately in a low pH (e.g., pH 4.0) buffer. The mass ratio of ionizable lipid to encapsulated nucleic acid is maintained within a desired range (e.g., 20:1 to 5:1). To form the lipid nanoparticle core, the nucleic acid in the low pH buffer is rapidly mixed with an ethanol solution in a desired volume ratio (e.g., 3:1). In this process, the low pH buffer positively charges the ionizable lipids, facilitating the encapsulation of the negatively charged oligonucleotides. After mixing, the nanoparticles are dialyzed against 1x PBS to remove ethanol and residual buffer. Finally, to form the LNP-SNA from the LNP, one or more oligonucleotides are attached to the exterior of the lipid nanoparticle core by mixing the LNP with the oligonucleotide and lipid-PEG conjugate containing the conjugation site in a desired ratio (e.g., 1:1).

[0057] In any of the aspects or embodiments of the present disclosure, the components of the lipid nanoparticle core include ionizable lipids, phospholipids, sterols, encapsulated oligonucleotides, and lipid-polyethylene glycol (lipid-PEG) conjugates. Various amounts of each component can be used to generate the lipid nanoparticle core. In some embodiments, the lipid nanoparticle core of the LNP-SNA includes a mole fraction of ionizable lipid that is about 50% of the total lipid in the LNP-SNA. In some embodiments, the ionizable lipid is dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA). In some embodiments, the lipid nanoparticle core of the LNP-SNA comprises a molar fraction of phospholipids that is between 1% and 25%, or between 2% and 5%, or between 5% and 20%, or between 10% and 25%, or between 10% and 20%, or between about 1% and about 25%, or between about 2% and about 5%, or between about 5% and about 20%, or between about 10% and about 25%, or between about 10% and about 20% of the total lipids in the LNP-SNA. In further embodiments, the lipid nanoparticle core of the LNP-SNA is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total lipid in the LNP-SNA. , 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%, or less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%. In some embodiments, the lipid nanoparticle core of the LNP-SNA comprises a molar fraction of phospholipids that is 3.5% or about 3.5% of the total lipid in the LNP-SNA.In various embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dihexadecanoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or a combination thereof. In some embodiments, the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the sterol is cholesterol. In some embodiments, the lipid nanoparticle core of the LNP-SNA comprises a molar fraction of sterol that is about 20% to about 50%, or about 25% to about 45%, or about 20% to about 35%, or about 20% to about 30%, or about 25% to about 35% of the total lipid in the LNP-SNA. In further embodiments, the lipid nanoparticle core of the LNP-SNA is at least about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of the total lipid in the LNP-SNA. , 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%, or less than about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%. In some embodiments, the lipid nanoparticle core of the LNP-SNA comprises a sterol mole fraction that is, or is at least about, or is less than about 45%. In some embodiments, the sterol is cholesterol.In some embodiments, the LNP-SNA comprises a mole fraction of cholesterol that is 45% or about 45% of the total lipids in the LNP-SNA. In some embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate comprises 2000 Dalton (Da) polyethylene glycol. In further embodiments, the lipid-polyethylene glycol (lipid-PEG) conjugate is a lipid-PEG-maleimide. In still further embodiments, the lipid-PEG-maleimide is 1,2-dipalmitryl-sn-glycero-3-phosphoethanolamine (DPPE) conjugated to a 2000 Da polyethylene glycol maleimide, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) conjugated to a 2000 Da polyethylene glycol maleimide, or a combination thereof. In some embodiments, the LNP-SNA comprises a molar fraction of lipid-PEG conjugate that is between about 1% and about 5%, or between about 1% and about 4%, or between about 1.5% and about 5%, or between about 1.5% and about 4%, or between about 1% and about 3.5%, or between about 1.5% and about 3%, or between about 1% and about 2%, or between about 1% and about 2.5% of the total lipid in the LNP-SNA. In further embodiments, the lipid nanoparticle core of the LNP-SNA comprises a mole fraction of lipid-PEG conjugate that is 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% of the total lipid in the LNP-SNA, or is at least about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% or is less than about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%. In some embodiments, the lipid nanoparticle core of the LNP-SNA comprises a mole fraction of lipid-PEG conjugate that is 1.5%, or is at least about 1.5%, or is less than about 1.5%. In some embodiments, the lipid-PEG conjugate is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) conjugated to 2000 Da polyethylene glycol maleimide. In some embodiments, the mass ratio between the ionizable lipid and the encapsulated oligonucleotide in the LNP-SNA is about 5:1.In further embodiments, the mass ratio of ionizable lipid to encapsulated oligonucleotide in the LNP-SNA is about 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

[0058] Use of LNP-SNAs in gene regulation / therapy In any of the aspects or embodiments of the present disclosure, it is contemplated that the LNP-SNA disclosed herein has the ability to regulate gene expression. Thus, in some embodiments, the LNP-SNA of the present disclosure comprises a lipid nanoparticle core and a shell of oligonucleotides bound to the exterior of the lipid nanoparticle core, the shell of oligonucleotides comprising one or more oligonucleotides having gene regulation activity (e.g., inhibition of target gene expression or target cell recognition). In some embodiments, the shell of oligonucleotides bound to the exterior of the lipid nanoparticle core of the LNP-SNA comprises one or more oligonucleotides that are inhibitory oligonucleotides as described herein. In some embodiments, the inhibitory oligonucleotides are encapsulated in the lipid nanoparticle core of the LNP-SNA. In some embodiments, the inhibitory oligonucleotides are encapsulated in the lipid nanoparticle core of the LNP-SNA, and the shell of oligonucleotides bound to the exterior of the lipid nanoparticle core of the LNP-SNA comprises one or more oligonucleotides that are inhibitory oligonucleotides. Thus, in some embodiments, the disclosure provides methods of inhibiting expression of a gene product, such methods comprising: inhibiting expression of a target gene product by about or at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% or less compared to expression of the gene product in the absence of LNP-SNA. or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least about 99%, or 100% inhibited.In other words, the methods provided encompass those that result in essentially any degree of inhibition of expression of the target gene product.

[0059] The degree of inhibition is determined in vivo from bodily fluid or biopsy samples, or by imaging techniques well known in the art, or alternatively, the degree of inhibition is determined in cell culture assays as a predictable measure of the degree of inhibition that can generally be expected in vivo resulting from the use of a particular type of LNP-SNA and a particular oligonucleotide.

[0060] Thus, there is provided a method of utilizing the LNP-SNA of the present disclosure in gene regulatory therapy, the method comprising hybridizing a target polynucleotide encoding a gene product with one or more oligonucleotides of the LNP-SNA that are complementary to all or a portion of the target polynucleotide, where hybridization between the target polynucleotide and the oligonucleotide occurs over the length of the target polynucleotide with a sufficient degree of complementarity to inhibit expression of the gene product. Inhibition of gene expression can occur in vivo or in vitro.

[0061] In various embodiments, the inhibitory oligonucleotides utilized in the methods of the present disclosure are RNA, DNA, or modified forms thereof. In various embodiments, the inhibitory oligonucleotides are antisense DNA, small interfering RNA (siRNA), aptamers, short hairpin RNA (shRNA), DNAzymes, or aptazymes.

[0062] Use of LNP-SNAs in immune modulation Toll-like receptors (TLRs) are a class of proteins expressed on sentinel cells and play a key role in regulating the innate immune system. The mammalian immune system uses two general strategies to fight infections. Exposure to pathogens rapidly triggers an innate immune response characterized by the production of immunostimulatory cytokines, chemokines, and polyreactive IgM antibodies. The innate immune system is activated by exposure to pathogen-associated molecular patterns (PAMPs) expressed by a diverse group of infectious microorganisms. Recognition of PAMPs is mediated by members of the Toll-like family of receptors. TLR receptors, such as TLR4, TLR8, and TLR9, which respond to specific oligonucleotides, reside within specialized intracellular compartments called endosomes. Regulatory mechanisms of TLR4, TLR8, and TLR9 receptors are based on, for example, but not limited to, DNA-protein interactions.

[0063] As described herein, synthetic immunostimulatory oligonucleotides containing CpG motifs similar to those found in bacterial DNA stimulate similar responses of TLR receptors. Thus, the CpG oligonucleotides of the present disclosure have the ability to function as TLR agonists. Other TLR agonists contemplated by the present disclosure include, but are not limited to, single-stranded RNA and small molecules (e.g., R848 (resiquimod)). Thus, immune-modulating oligonucleotides have a variety of promising therapeutic applications, such as the treatment of immune deficiencies and cancer. Thus, in some embodiments, the LNP-SNAs of the present disclosure are used in methods to modulate the activity of toll-like receptors (TLRs).

[0064] In further embodiments, the LNP-SNA of the present disclosure comprises an oligonucleotide that is a TLR antagonist. In some embodiments, the TLR antagonist is single-stranded DNA (ssDNA).

[0065] In some embodiments, the downregulation of the immune system involves knocking down the genes involved in the expression of Toll-like receptors. This antisense approach involves the use of the LNP-SNA of the present disclosure to inhibit the expression of any toll-like protein.

[0066] Thus, in some embodiments, methods are disclosed that utilize the LNP-SNA described herein to modulate toll-like receptors. The methods use a TLR agonist or TLR antagonist to either up-regulate or down-regulate toll-like receptor activity, respectively. The methods include contacting a cell having a toll-like receptor with the LNP-SNA of the present disclosure, thereby modulating the activity and / or expression of the toll-like receptor. The toll-like receptors that are modulated include one or more of toll-like receptor 1, toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 11, toll-like receptor 12, and / or toll-like receptor 13.

[0067] Use of LNP-SNA to treat disorders In some embodiments, the LNP-SNA of the present disclosure is used to treat a disorder. As used herein, "treat" or "treating" means to eliminate, reduce, or ameliorate a disorder or one or more symptoms thereof. Thus, in some aspects, the present disclosure provides a method of treating a disorder, comprising administering an effective amount of the LNP-SNA of the present disclosure to a subject (e.g., a human subject) in need thereof, thereby treating the disorder. In various embodiments, the disorder is cancer, an infectious disease, an autoimmune disease, or a combination thereof. An "effective amount" of the LNP-SNA is, for example, an amount sufficient to affect gene editing, inhibit gene expression, and / or activate an innate immune response. Thus, methods of activating an innate immune response are also contemplated herein, including administering an LNP-SNA of the present disclosure to a subject in need thereof in an amount effective to activate an innate immune response in the subject.

[0068] The LNP-SNA of the present disclosure can be administered via any suitable route, such as parenteral administration, intramuscular injection, subcutaneous injection, intradermal administration, and / or mucosal administration, such as oral or intranasal. Additional routes of administration include, but are not limited to, intravenous, intraperitoneal, intranasal, intravaginal, intrarectal, and oral administration. Combinations of different routes of administration, either separately or simultaneously, are also contemplated by the present disclosure.

[0069] Use of LNP-SNA in detection In some embodiments, the LNP-SNAs of the present disclosure are useful in nanoflare technology. Nanoflare has been previously described in the context of polynucleotide-functionalized nanoparticles for fluorescent detection of target molecule levels in living cells (described in U.S. Patent Application Publication No. 2010 / 0129808, which is incorporated herein by reference in its entirety). In this system, the "flare" is detectably labeled, and in some embodiments, is a single strand (or a portion of a single strand oligonucleotide) of a double-stranded oligonucleotide, labeled with a detectable marker, and displaced or released from the LNP-SNA by an incoming target polynucleotide. Thus, it is contemplated that nanoflare technology is useful in the context of the LNP-SNAs described herein.

[0070] composition The present disclosure also provides a composition comprising the LNP-SNA of the present disclosure, or a plurality thereof. In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier. The term "carrier" refers to a vehicle in which the SNA described herein is administered to a subject. Any conventional medium or agent compatible with the LNP-SNA according to the present disclosure can be used. The term carrier includes diluents, excipients, adjuvants, and combinations thereof.

[0071] Therapeutic Agents The LNP-SNA provided herein optionally further comprises a therapeutic agent or agents. In various embodiments, the therapeutic agent is simply associated with an oligonucleotide within the shell of an oligonucleotide bound to the exterior of the lipid nanoparticle core of the LNP-SNA, and / or the therapeutic agent is associated with the lipid nanoparticle core of the LNP-SNA, and / or the therapeutic agent is encapsulated in the lipid nanoparticle core of the LNP-SNA. In some embodiments, the therapeutic agent is associated with the end of an oligonucleotide within the shell of an oligonucleotide that is not bound to the lipid nanoparticle core (e.g., if the oligonucleotide is bound to the lipid nanoparticle core via its 3' end, the therapeutic agent is associated with the 5' end of the oligonucleotide). Alternatively, in some embodiments, the therapeutic agent is associated with the end of an oligonucleotide within the shell of an oligonucleotide that is bound to the lipid nanoparticle core (e.g., if the oligonucleotide is bound to the lipid nanoparticle core via its 3' end, the therapeutic agent is associated with the 3' end of the oligonucleotide). In some embodiments, the therapeutic agent is covalently associated with an oligonucleotide within the shell of an oligonucleotide bound to the exterior of the lipid nanoparticle core of the LNP-SNA. In some embodiments, the therapeutic agent is non-covalently associated with the oligonucleotides within the shell of oligonucleotides bound to the exterior of the lipid nanoparticle core of the LNP-SNA. However, it should be understood that the present disclosure provides LNP-SNAs in which one or more therapeutic agents are both covalently and non-covalently associated with the oligonucleotides within the shell of oligonucleotides bound to the exterior of the lipid nanoparticle core of the LNP-SNA. It should also be understood that non-covalent association includes hybridization, protein binding, and / or hydrophobic interactions. In some embodiments, the therapeutic agent is administered separately from the LNP-SNA of the present disclosure. Thus, in some embodiments, the therapeutic agent is administered before, after, or simultaneously with the LNP-SNA of the present disclosure to treat a disorder.

[0072] Therapeutic agents contemplated by the present disclosure include, but are not limited to, a protein (e.g., a therapeutic protein), a growth factor, a hormone, an interferon, an interleukin, an antibody or antibody fragment, a small molecule, a peptide, an antibiotic, an antifungal agent, an antiviral agent, a chemotherapeutic agent, or a combination thereof.

[0073] The term "small molecule" as used herein refers to a chemical compound or drug, or any other low molecular weight organic compound, either natural or synthetic. By "low molecular weight" we mean a compound having a molecular weight of less than 1500 Daltons, typically between 100 and 700 Daltons.

[0074] Detectable Markers In any of the aspects or embodiments of the present disclosure, the oligonucleotides (e.g., one or more oligonucleotides within a shell of oligonucleotides bound to the exterior of the lipid nanoparticle core of the LNP-SNA and / or one or more oligonucleotides encapsulated in the lipid nanoparticle core of the LNP-SNA) comprise a detectable marker (e.g., a fluorophore and / or a radioactive label). Methods for binding detectable markers to oligonucleotides or nanoparticle cores are known in the art.

[0075] In some embodiments, detectable marker is associated with lipid nanoparticle core.For example, but not limited to, lipid nanoparticle core of the present disclosure can be labeled with fluorophore.In some embodiments, both lipid nanoparticle core, one or more oligonucleotides bound to the exterior of lipid nanoparticle core, and / or one or more oligonucleotides encapsulated within lipid nanoparticle core contain fluorophore, and the fluorophores can all be the same, or one or more fluorophores can be different. 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[0076] The following examples are provided merely to illustrate the present disclosure and not to limit its scope. EXAMPLES

[0077] Example 1 To form lipid nanoparticles (LNPs), the components of the structures were dissolved in ethanol at a total concentration of 20–80 mM. These components are divided into four different classes: ionizable lipids, phospholipids, sterols, and lipid-PEG-maleimide. Each nanoparticle structure was composed of one component from each class. The mole fractions of each component were: ionizable lipids, 50%; phospholipids, 1.4–23.5%; sterols, 25–45%; and lipid-PEG-maleimide, 1.5–3.5%. The ionizable lipid used was dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA). The phospholipids used were 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). The sterol used was cholesterol. The lipid-PEG-maleimides used were 1,2-dipalmitryl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) conjugated to a 2000 Da polyethylene glycol maleimide. The nucleic acid to be encapsulated was dissolved in 10 mM sodium citrate buffer at pH 4.0. The mass ratio used between the ionizable lipid and the nucleic acid used was 5:1. To encapsulate the nucleic acid, the nucleic acid was mixed with the LNP components in ethanol in a volume ratio of 3:1 using a pipette tip (Figure 1). After mixing, the LNP was dialyzed against phosphate buffered saline in a 3000 Da molecular weight cutoff membrane for 2 hours.

[0078] Lipid nanoparticle spherical nucleic acids (LNP-SNAs) were formed from conjugating sulfhydryl-terminated DNA sequences to lipid-PEG-maleimide on the surface of dialyzed LNPs. DNA synthesized on 1-O-dimethoxytrityl-propyl-disulfide was conjugated to 1'-succinyl-lcaa-CPG by reduction to form a 3' sulfhydryl group using 100 mM 1,4-dithiothreitol (DTT) at pH 8.3-8.5.

[0079] After removing DTT using a Sephadex G-25 column, 1 equivalent of DNA was mixed with LNP and shaken at room temperature for 2 h to form LNP-SNA in phosphate-buffered saline at pH 7.2–7.6. The diameter and structure of LNP-SNA were confirmed using nanoparticle tracking analysis and cryo-electron microscopy (Figure 2).

[0080] After reaction with LNP, DNA mobility was reduced on an agarose gel to confirm conjugation (Figure 3), and LNP-SNA activity was evaluated in relation to dsDNA and siRNA delivery in cellular assays. In the Raw264.7 cell line, designed to respond to activation of the cGAS-STING pathway, LNP-SNA showed a critical concentration of 28.1 nM compared to 2'3'-cGAMP (4.75 µM) and free DNA, which showed no activation (Figure 4A). In a U87 cell line expressing luciferase (Luc2), siLuc2 delivery was used as a proof of concept for LNP-SNA-mediated gene silencing. LNP-SNA containing siLuc2 silenced Luc2 expression by approximately 90% at concentrations of 25-50 nM compared to siGFP control LNP-SNA (Figure 4B). Compared to comparable LNP constructs, LNP-SNA silenced approximately 5% more Luc2 at concentrations of 50 nM and 100 nM in the U87-Luc2 cell line (FIG. 4C).

[0081] The function and targeting of LNP-SNA was evaluated in C57BL / 6 mice. LNP-SNA and comparable LNP were formulated with luciferase mRNA. Luminescence was assessed 6 hours after 0.1 mg kg-1 injection of mRNA via the tail vein. LNP-SNA showed spleen-specific mRNA expression with no detectable expression in the liver (Figure 5). In contrast, LNP showed a high degree of mRNA expression in the liver and a similar level of expression as LNP-SNA in the spleen, indicating a greater potential for off-target hepatotoxicity. Figure 5 shows that the presence of DNA sequences on the surface of the SNA alters where mRNA expression is observed. In addition to altering the delivery of comparable LNPs, delivery can be tuned by altering the sequence composition of the surface DNA. Figure 6 shows that there is a significant difference in where mRNA expression is achieved between LNP-SNA with surface G-rich DNA sequences and LNP-SNA with T-rich DNA sequences.

[0082] Materials and Methods DNA was synthesized using automated solid-support phosphoramidite synthesis (model: MM12, BioAutomation, Inc.). Sequences were purified by reversed-phase high-performance liquid chromatography (HPLC, Agilent Technologies) and characterized using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-ToF, Bruker Autoflex III). The DNA sequences and lipid nanoparticle compositions used in the experiments are listed in Table 1 below. Firefly luciferase mRNA was purchased from TriLink BioTechnologies.

[0083] DLin-MC3-DMA was purchased from MedChemExpress. DMPE-PEG(2000)maleimide, DPPE-PEG(2000)maleimide, and DSPE-PEG(2000)maleimide were purchased from Nanocs, Inc. Cholesterol and Triton™-X-100 were purchased from Sigma. DOPC, DSPC, 18:1DAP, and DOPE were purchased from Avanti Polar lipidds, Inc. Lipofectamine™ 2000, Quant-iT™ PicoGreen™ dsDNA reagent, Quant-iT™ RiboGreen™ reagent, and 20x TE buffer were purchased from ThermoFisher. D-Luciferin was purchased from Gold BioTechnologies, and Luc mRNA was purchased from TriLink BioTechnologies.

[0084] LNP-SNA formulation. LNPs were formulated using the ethanol dilution method [Cheng et al., Dendrimer-Based Lipid Nanoparticles Deliver Therapeutic FAH mRNA to Normalize Liver Function and Extend Survival in a Mouse Model of Hepatorenal Tyrosinemia Type I. Advanced Materials 30(52):e1805308(2018)]. Briefly, lipids and cholesterol were dissolved in 100% ethanol. dsDNA was dissolved in 10 mM citrate at pH 4.0 with a mass ratio of ionizable lipid:dsDNA of 5.0. After making both solutions, DNA was rapidly pipette mixed with the ethanol solution at a volume ratio of 3:1. After mixing, NPs were dialyzed twice against 1×PBS for 60 min in a Pierce™ 3K MWCO microdialysis plate (ThermoFisher). Subsequently, NPs were added to a microcentrifuge tube containing one equivalent of lyophilized thiol-terminated DNA sequence and shaken at 700 rpm at room temperature overnight to facilitate the reaction of the maleimide-functionalized PEG lipid with the sulfhydryl-terminated DNA.

[0085] Characterization of LNP-SNA. LNP-SNA size and nanoparticle concentration were determined by nanoparticle tracking analysis (NTA) using a Malvern NanoSight NS300 equipped with NanoSight sample assistant. Nanoparticles were diluted 1:1000 in water and passed through the microfluidics at 50 μL / min. Size was determined using NTA software with detection thresholds set manually to avoid background. Encapsulation efficiency of dsDNA and RNA was determined by modified Quant-iT™ PicoGreen™ and Quant-iT™ RiboGreen™ (Invitrogen) assays, respectively. Briefly, two separate standard curves were generated with the encapsulated nucleic acids. One was in 1×TE buffer and the other was in 1×TE supplemented with 0.1% Triton™-X-100. Two samples were made from each nanoparticle, one diluted in TE and one diluted in TE with 0.1% Triton™-X-100. 100 μL of 1× PicoGreen™ (dsDNA) or RiboGreen™ was then added on top of the standards and samples, and the fluorescence of each sample was measured using a plate reader. The concentration of free nucleic acid was determined from the TE standard curve, and the concentration of total nucleic acid was determined by dissolving the particles in 0.1% Triton™-X-100. From this, the encapsulation efficiency was calculated from the following formula: ([Triton-X]-[TE]) / ([Triton-X]) or ([Total]-[Free]) / ([Total]).

[0086] Cellular assays for measuring cGAS-STING pathway activation. Raw Lucia™ ISG (Raw264.7) cell line was purchased from Invivogen. For in vitro experiments, Zeocin™, Normocin™, and QUANTI-Luc™ were also purchased from Invivogen. All cell lines were cultured according to the manufacturer's specifications. No cell authentication was performed. All cell lines were tested for mycoplasma contamination and cultured at 37° C. with 5% CO 2The bacteria were grown in a humidified atmosphere at 20 °C.

[0087] Certain nanoparticle formulations and controls were diluted in Opti-MEM (Gibco) and plated in triplicate in 96-well plates. Cells were then plated on top of the nanoparticle treatments at 100,000 cells per well. After 24 hours of incubation, 20 μL of medium was removed and IRF3 induction was quantified using Quanti-Luc™ reagent (Invivogen) according to the manufacturer's protocol. PrestoBlue™ HS cell permeable viability reagent (Thermo Fisher) was used to normalize the number of viable cells to the amount of IRF3 induction achieved. After removing 20 μL of medium for Quanti-Luc™ measurements, additional medium was removed so that the volume in the plate was 90 μL. 10 μL of PrestoBlue™ was added per well and the plate was incubated for 15 minutes, at which point fluorescence was read according to the manufacturer's protocol. IRF3 induction (luminescence) was then normalized to viable cells (PrestoBlue™ fluorescence) on a per-well basis.

[0088] LNP-SNA delivering siRNA in cell assays. B16-F10-Luc2 and U87-Luc2 cell lines were obtained from ATCC and cultured according to the manufacturer's specifications. To evaluate siRNA-mediated gene silencing, the top five LNP-SNA candidates from the cGAS-STING pathway screen were formulated with siLuc2 in combination with a control LNP-SNA formulated with siGFP. Thus, gene silencing can be read out as a decrease in luminescence due to silencing of Luc2.

[0089] The specific nanoparticle formulations and transfected siRNA controls were diluted in Opti-MEM (Gibco) and plated in triplicate in 96-well plates. Cells were then plated on top of the nanoparticle treatments at 50,000 cells per well. After 24 hours of incubation, 120 μL of medium was removed and 20 μL of CellTiter-Fluor™ reagent (Promega) was added to measure the number of viable cells in each well. After 30 minutes of incubation at 37°C, fluorescence was read according to the manufacturer's protocol. The wells were then washed three times with 100 uL of PBS. Luc2 luminescence was read using a luciferase assay system (Promega). Luc2 gene silencing was assessed in arbitrary units normalized to CellTiter-Fluor™ viability.

[0090] Animal handling. Female mice (C57Bl / 6) ranging in age from 8 to 12 weeks were obtained from Jackson Laboratory and maintained in a conventional breeding facility. All animals used were handled according to methods and procedures approved by the Institutional Animal Care and Use Committee of Northwestern University under protocol IS00010970.

[0091] Luciferase (Luc2) mRNA expression. Cleancap® luciferase mRNA was purchased from TriLink Biotechnologies. Mice were administered 0.1 mg kg -1 Six hours later, mice were given a single bolus injection of the mRNA-containing formulation at 150 mg kg -1 D-luciferin was injected intraperitoneally. The animals were then euthanized and the major organs were harvested and immersed in 300 μg / mL D-luciferin solution. Individual organs were then imaged using an IVIS Spectrum instrument (Perkin Elmer).

[0092] [Table 1-1] [Table 1-2] [Table 1-3] The percentages listed in Table 1 represent the amounts (in mole percent) of the various components in the final LNP-SNA. C14, C16 and C18 are the lengths of the lipid alkyl chains. The lipids are named as follows: DMPE-PEG-maleimide (C14), DPPE-PEG-maleimide (C16), and DSPE-PEG-maleimide (C18). "T21" refers to the sequence 5'-TTTTTTTTTT TTTTTTTTTT T-3' (SEQ ID NO:1). "GGT7" refers to the sequence 5'-GGTGGTGGTG GTGGTGGTGG T-3' (SEQ ID NO:2). "EE%" refers to encapsulation efficiency.

[0093] Example 2 Ai14 mouse experiments General animal handling and isolation protocols for specific cell types Animal handling. Female mice (C57Bl / 6J (#000664) and LSL-Tomato / Ai14 (#007914)) ranging in age from 8 to 12 weeks were obtained from Jackson Laboratory and maintained in a conventional breeding facility. All animals used were handled according to methods and procedures approved by the Animal Care and Use Committee of Northwestern University.

[0094] Cell isolation. Organs were harvested and incubated in a digestion mixture of 5000 U / mL collagenase I for 30 min at 37°C. Organs were then minced into small slices approximately 3 mm thick and passed through a 70 μm filter. Red blood cells were then lysed using ACK lysis buffer (Thermo-Fisher) for 5 min, and cells were counted and resuspended in PBS containing 2.5% bovine serum albumin.

[0095] The cell types of interest from each organ were isolated using magnetic separation. Splenic macrophages and B cells were isolated using the EasySep™ kit (Stemcell™ Technologies). Liver B cells were isolated using the same EasySep™ kit. Hepatocytes were isolated by centrifugation at 200×g.

[0096] Ai14 mice: Ai14 mice were transfected with Cre mRNA at 0.3 mg / kg -1 LNP and LNP-SNA were injected intravenously at a dose of 0.01 mg / kg / day (Figure 7). Figure 7 shows an assay for cell population level genome editing using Ai14 mice. Ai14 mice express tdTom downstream of a floxed stop cassette (Figure 7A). Expression of Cre recombinase removes the stop cassette and turns on tdTom expression. This can be detected via flow cytometry, where the percentage of the target cell population expressing tdTom above background is quantified. This is displayed as % tdTom+ cells.

[0097] Two days after injection, the animals were euthanized and the cell types of interest were isolated using the cell isolation protocol described above. Once the individual cell populations were prepared, the cells were run on a flow cytometer. Gated on PBS-treated mice, genome editing in each cell type was quantified in terms of the percent of cells with detectable tdTomato fluorescence (Figure 8). Figure 9 shows that LNP-SNAs functionalized with the GGT sequence induce genome editing by Cre mRNA in splenic monocytes.

[0098] Regarding DNA barcoding experiments, Synthesis and administration of LNP-SNA in C57BL / 6J mice. Small libraries of LNP-SNA and naked LNP were generated with the compositions shown in Table 2 below. Each LNP or LNP-SNA encapsulated a unique 56 base DNA barcode that identifies each particle. After quantifying the amount of barcode within each particle, equal amounts of barcode were added at 0.1 mg kg -1The cells were pooled into a total dose of 1000 μg / ml and injected into C57BL / 6J mice. After a 2-day circulation period, the cell types of interest were isolated by magnetic separation using the protocol above and proceeded to DNA isolation and sequencing (described below).

[0099] [Table 2]

[0100] DNA isolation and next generation sequencing. For cell types of interest, DNA was isolated using Clarity OTX™ columns (Phenomenex). Samples were lyophilized and cleaned using the PCR-Cleanup kit (New England Biolabs, Inc.). Nested PCR was then performed according to previous protocols [Paunovska, K.; Sago, CD; Monaco, CM; Hudson, WH; Castro, MG; Rudoltz, TG; Kalathoor, S.; Vanover, DA; Santangelo, PJ; Ahmed, R.; Bryksin, AV; Dahlman, JEA Direct Comparison of in Vitro and in Vivo Nucleic Acid Delivery Mediated by Hundreds of Nanoparticles Reveals a Weak Correlation. Nano Lett. 2018, 18(3), 2148-2157. https: / / doi.org / 10.1021 / acs.nanolett.8b00432]. Barcode sequences were amplified using universal primers, followed by sample indexing using adapter sequences and addition of Nextera XT chemistry. Samples were sequenced using an Illumina NextSeq™.

[0101] Analysis of NGS data. Sequencing files were analyzed using custom R scripts. First, reads were preprocessed to exclude reads shorter than 40 bases as well as adapter primer sequences. Next, reads were processed such that no reads contained a quality score below 20. Finally, each barcode was counted within each sample by searching for the reverse complement of the barcode sequence. The number of reads for each barcode in each sample was used to normalize the numbers to the input. This was used to normalize the number of reads from each barcode to the number originally injected. Delivery was then quantified as "normalized delivery" or the percentage of normalized reads from each barcode as a percentage of the total number of reads in the sample.

[0102] FIG. 10 shows that the (GGT)7 outer sequence and DOPE helper lipid enabled enhanced delivery of LNP-SNAs to the major splenic cell types. Delivery to the spleen was assessed using DNA barcoding technology. LNP-SNAs were indexed with 56 nucleotide-long barcodes and amplified and indexed using the strategy shown in the left panel of FIG. 10. Delivery of LNP and LNP-SNA constructs was assessed as the percentage of total barcode reads derived from each cell type of interest. Here, it was found that enrichment of LNP-SNAs in the spleen was achieved by presenting the (GGT)7 outer sequence and containing the helper lipid DOPE.

Claims

1. A lipid nanoparticle spherical nucleic acid (LNP-SNA) comprising a lipid nanoparticle core and an oligonucleotide shell composed of oligonucleotides attached to the exterior of the lipid nanoparticle core, the lipid nanoparticle core comprises an encapsulated oligonucleotide, an ionizable lipid, a phospholipid, a sterol, and a lipid-polyethylene glycol (lipid-PEG) conjugate; A LNP-SNA, wherein one oligonucleotide within the oligonucleotide shell is covalently attached to the exterior of the lipid nanoparticle core via the lipid-PEG conjugate.

2. The LNP-SNA of claim 1, wherein at least 10% of the oligonucleotides within the oligonucleotide shell are covalently bound to the exterior of the lipid nanoparticle core via the lipid-PEG conjugate.

3. The LNP-SNA of claim 1 or 2, wherein the shell of oligonucleotides comprises from about 5 to about 1000 oligonucleotides.

4. The LNP-SNA of any one of claims 1 to 3, wherein the shell of oligonucleotides comprises from about 100 to about 1000 oligonucleotides.

5. The LNP-SNA according to any one of claims 1 to 4, wherein the shell of oligonucleotides comprises about 400 oligonucleotides.

6. The LNP-SNA of any one of claims 1 to 5, wherein each oligonucleotide within said shell of oligonucleotides is from about 5 to about 100 nucleotides in length.

7. The LNP-SNA of any one of claims 1 to 6, wherein each oligonucleotide within said shell of oligonucleotides is from about 10 to about 50 nucleotides in length.

8. The LNP-SNA of any one of claims 1 to 7, wherein each oligonucleotide within the shell of oligonucleotides is about 25 nucleotides in length.

9. The LNP-SNA of any one of claims 1 to 8, wherein each oligonucleotide within the shell of oligonucleotides has the same nucleotide sequence.

10. The LNP-SNA of any one of claims 1 to 8, wherein the shell of oligonucleotides comprises at least two oligonucleotides having different nucleotide sequences.

11. The LNP-SNA of any one of claims 1 to 10, wherein the shell of oligonucleotides is composed of single-stranded, double-stranded DNA oligonucleotides, or a combination thereof.

12. The LNP-SNA of any one of claims 1 to 10, wherein the shell of oligonucleotides is composed of single-stranded, double-stranded RNA oligonucleotides, or a combination thereof.

13. The LNP-SNA of any one of claims 1 to 10, wherein the shell of oligonucleotides is composed of single-stranded DNA oligonucleotides, double-stranded DNA oligonucleotides, single-stranded RNA oligonucleotides, double-stranded RNA oligonucleotides, or combinations thereof.

14. The LNP-SNA of any one of claims 1 to 13, wherein at least one oligonucleotide within the shell of oligonucleotides is a targeting oligonucleotide.

15. The LNP-SNA of any one of claims 1 to 14, wherein each oligonucleotide within the shell of oligonucleotides is a targeting oligonucleotide.

16. At least one oligonucleotide within the shell of oligonucleotides is (GGT) ｎ 16. The LNP-SNA of any one of claims 1 to 15, comprising or consisting of a nucleotide sequence, wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

17. Each oligonucleotide within the shell of oligonucleotides is (GGT) ｎ 17. The LNP-SNA of any one of claims 1 to 16, comprising or consisting of a nucleotide sequence, wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

18. The LNP-SNA of claim 16 or 17, wherein n is 7.

19. The LNP-SNA of any one of claims 1 to 18, wherein at least one oligonucleotide within the shell of oligonucleotides is an aptamer.

20. The LNP-SNA of any one of claims 1 to 19, wherein at least one oligonucleotide within the shell of oligonucleotides comprises a detectable marker.

21. The LNP-SNA of any one of claims 1 to 20, wherein the shell of oligonucleotides comprises an inhibitory oligonucleotide, an immunostimulatory oligonucleotide, a gene editor substrate DNA or RNA, or a combination thereof.

22. The LNP-SNA of claim 21, wherein the inhibitory oligonucleotide is an antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme.

23. The LNP-SNA of claim 21 or 22, wherein the immunostimulatory oligonucleotide is a CpG motif-containing oligonucleotide, a double-stranded DNA oligonucleotide, or a single-stranded RNA oligonucleotide.

24. The LNP-SNA of any one of claims 1 to 23, wherein the encapsulated oligonucleotide is composed of DNA, RNA, or a combination thereof.

25. The LNP-SNA of any one of claims 1 to 24, wherein the encapsulated oligonucleotide is an inhibitory oligonucleotide, an mRNA, an immunostimulatory oligonucleotide, an mRNA encoding a gene editor protein, or a DNA or RNA gene editor substrate.

26. The LNP-SNA of claim 25, wherein the inhibitory oligonucleotide is an antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme.

27. The LNP-SNA of claim 25 or 26, wherein the immunostimulatory oligonucleotide is a CpG motif-containing oligonucleotide.

28. The LNP-SNA of any one of claims 1 to 27, wherein the encapsulated oligonucleotide is from about 5 to about 5000 nucleotides in length.

29. The LNP-SNA of any one of claims 1 to 28, wherein the encapsulated oligonucleotide is from about 10 to about 4500 nucleotides in length.

30. The LNP-SNA of any one of claims 1 to 29, wherein the encapsulated oligonucleotide is about 1500 nucleotides in length.

31. The LNP-SNA of any one of claims 1 to 30, wherein the lipid nanoparticle core comprises a plurality of encapsulated oligonucleotides.

32. 32. The LNP-SNA of claim 31, wherein at least one oligonucleotide in said plurality of encapsulated oligonucleotides comprises a detectable marker.

33. The LNP-SNA of claim 31 or 32, wherein the plurality of encapsulated oligonucleotides comprises an inhibitory oligonucleotide, an mRNA, an immunostimulatory oligonucleotide, an mRNA encoding a gene editor protein, a DNA or RNA gene editor substrate, or a combination thereof.

34. The LNP-SNA of claim 33, wherein the inhibitory oligonucleotide is an antisense oligonucleotide, a small interfering RNA (siRNA), an aptamer, a short hairpin RNA (shRNA), a DNAzyme, or an aptazyme.

35. The LNP-SNA of claim 33 or 34, wherein the immunostimulatory oligonucleotide is a CpG motif-containing oligonucleotide, double-stranded DNA (dsDNA), double-stranded RNA, or single-stranded RNA (ssRNA).

36. The LNP-SNA of any one of claims 31 to 35, wherein each oligonucleotide in said plurality of encapsulated oligonucleotides is from about 10 to about 50 nucleotides in length.

37. The LNP-SNA of any one of claims 31 to 36, wherein each oligonucleotide in the plurality of encapsulated oligonucleotides is about 50 nucleotides in length.

38. The LNP-SNA of any one of claims 31 to 37, wherein each oligonucleotide in the plurality of encapsulated oligonucleotides has the same nucleotide sequence.

39. The LNP-SNA of any one of claims 31 to 37, wherein the plurality of encapsulated oligonucleotides comprises at least two oligonucleotides having different nucleotide sequences.

40. The LNP-SNA of any one of claims 1 to 39, wherein the ionizable lipid is dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), C12-200, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), or a combination thereof.

41. The LNP-SNA of any one of claims 1 to 40, wherein the ionizable lipid is dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA).

42. The LNP-SNA of any one of claims 1 to 41, wherein the LNP-SNA comprises a molar fraction of the ionizable lipid that is about 50% of the total lipid in the LNP-SNA.

43. The LNP-SNA according to any one of claims 1 to 42, wherein the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dihexadecanoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or a combination thereof.

44. The LNP-SNA of any one of claims 1 to 43, wherein the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

45. The LNP-SNA of any one of claims 1 to 44, wherein the LNP-SNA comprises a molar fraction of the phospholipid that is about 1% to about 25% of the total lipid in the LNP-SNA.

46. The LNP-SNA of any one of claims 1 to 45, wherein the LNP-SNA comprises a molar fraction of phospholipids that is 3.5% or about 3.5% of the total lipids in the LNP-SNA.

47. The sterols are 3β-hydroxycholest-5-ene (cholesterol), 9,10-secocholesta-5,7,10(19)-trien-3β-ol (vitamin D3), 9,10-secoegosta-5,7,10(19),22-tetraen-3β-ol (vitamin D2), calcipotriol, 24-ethyl-5,22-cholestadien-3β-ol (stigmasterol), 22,23-dihydrostigmasterol (β-sitosterol), 3,28-dihydroxy-lupeol (betulin), lupeol, ursolic acid, , oleanolic acid, 24α-methylcholesterol (campesterol), 24-ethylcholesta-5,24(28)E-dien-3β-ol (fucosterol), 24-methylcholesta-5,22-dien-3β-ol (brassicasterol), 24-methylcholesta-5,7,22-trien-3β-ol (ergosterol), 9,11-dehydroergosterol, daucosterol, or any of the aforementioned sterols modified with one or more amino acids.

48. The LNP-SNA of any one of claims 1 to 47, wherein the LNP-SNA comprises a molar fraction of the sterol that is about 25% to about 45% of the total lipids in the LNP-SNA.

49. The LNP-SNA of any one of claims 1 to 48, wherein the LNP-SNA comprises a molar fraction of sterol that is 45% or about 45% of the total lipids in the LNP-SNA.

50. The LNP-SNA of any one of claims 1 to 49, wherein the sterol is cholesterol.

51. 51. The LNP-SNA of claim 50, wherein the LNP-SNA comprises a molar fraction of cholesterol that is 45% or about 45% of the total lipids in the LNP-SNA.

52. The LNP-SNA of any one of claims 1 to 51, wherein the lipid-polyethylene glycol (lipid-PEG) conjugate comprises polyethylene glycol of 2000 Daltons (Da).

53. The LNP-SNA of any one of claims 1 to 52, wherein the lipid-polyethylene glycol (lipid-PEG) conjugate is lipid-PEG-maleimide.

54. 54. The LNP-SNA of claim 53, wherein the lipid-PEG-maleimide is 1,2-dipalmitryl-sn-glycero-3-phosphoethanolamine (DPPE) conjugated to a 2000 Da polyethylene glycol maleimide, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) conjugated to a 2000 Da polyethylene glycol maleimide, or a combination thereof.

55. The LNP-SNA of any one of claims 1 to 54, wherein the LNP-SNA comprises a molar fraction of the lipid-PEG conjugate that is about 1.5% to about 3.5% of the total lipid in the LNP-SNA.

56. The LNP-SNA of any one of claims 1 to 55, wherein the LNP-SNA comprises a molar fraction of the lipid-PEG conjugate that is 1.5% or about 1.5% of the total lipid in the LNP-SNA.

57. The LNP-SNA according to any one of claims 1 to 56, wherein the mass ratio between said ionizable lipid and said encapsulated oligonucleotide is from about 20:1 to about 5:

1.

58. The LNP-SNA of any one of claims 1 to 57, further comprising a therapeutic agent encapsulated in the lipid nanoparticle core.

59. The LNP-SNA of any one of claims 1 to 58, further comprising a therapeutic agent bound to the exterior of the lipid nanoparticle core.

60. The LNP-SNA of claim 58 or 59, wherein the therapeutic agent is an antibody or antibody fragment, a small molecule, a peptide, an antibiotic, a growth factor, a hormone, an interferon, an interleukin, an antifungal agent, an antiviral agent, a chemotherapeutic agent, or a combination thereof.

61. The LNP-SNA of any one of claims 1 to 60, further comprising a targeting peptide, a targeting antibody, or a combination thereof attached to the exterior of the lipid nanoparticle core.

62. A composition comprising a plurality of lipid nanoparticle spherical nucleic acid (LNP-SNA) molecules according to any one of claims 1 to 61.

63. 63. The composition of claim 62, further comprising a therapeutic agent.

64. 64. A composition as described in claim 62 or 63 for inhibiting expression of a gene by hybridizing to a polynucleotide encoding a gene product, wherein hybridization between the polynucleotide and one or more oligonucleotides within the shell of oligonucleotides occurs over the length of the polynucleotide with a sufficient degree of complementarity to inhibit expression of the gene product.

65. 64. A method of inhibiting expression of a gene in vitro comprising hybridizing a polynucleotide encoding a gene product with a lipid nanoparticle spherical nucleic acid (LNP-SNA) according to any one of claims 1 to 61, or a composition according to claim 62 or 63, wherein hybridization between the polynucleotide and one or more oligonucleotides within the shell of the oligonucleotide occurs over the length of the polynucleotide with a sufficient degree of complementarity to inhibit expression of the gene product. Method of posting.

66. 64. The composition of claim 62 or 63 for upregulating the activity of a toll-like receptor (TLR), wherein the composition is contacted with a cell that has the toll-like receptor.

67. 67. The composition of claim 66, wherein the shell of oligonucleotides comprises one or more oligonucleotides that are TLR agonists.

68. 68. The composition of claim 66 or 67, wherein the toll-like receptor is toll-like receptor 1, toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 11, toll-like receptor 12, toll-like receptor 13, or a combination thereof.

69. 64. The composition of claim 62 or 63 for down-regulating the activity of a toll-like receptor (TLR), wherein the composition is contacted with a cell that has the toll-like receptor.

70. 70. The composition of claim 69, wherein the shell of oligonucleotides comprises one or more oligonucleotides that are TLR antagonists.

71. 71. The composition of claim 69 or 70, wherein the toll-like receptor is toll-like receptor 1, toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 11, toll-like receptor 12, toll-like receptor 13, or a combination thereof.

72. A method for upregulating activity of a toll-like receptor (TLR) in vitro, comprising contacting a cell having the toll-like receptor with a lipid nanoparticle spherical nucleic acid (LNP-SNA) according to any one of claims 1 to 61, or a composition according to claim 62 or 63.

73. A method for down-regulating activity of a toll-like receptor (TLR) in vitro, comprising contacting a cell having the toll-like receptor with a lipid nanoparticle spherical nucleic acid (LNP-SNA) according to any one of claims 1 to 61, or a composition according to claim 62 or 63.

74. 64. A composition according to claim 62 or 63 for treating a disorder, wherein the disorder is treated by administering an effective amount of the composition to a subject in need thereof.

75. 75. The composition of claim 74, wherein the disorder is cancer, an infectious disease, an autoimmune disease, or a combination thereof.

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