WO2018067302A2 - Effets thérapeutiques de l'administration cellulaire de petites molécules et de macromolécules avec des acides nucléiques sphériques liposomaux - Google Patents

Effets thérapeutiques de l'administration cellulaire de petites molécules et de macromolécules avec des acides nucléiques sphériques liposomaux Download PDF

Info

Publication number
WO2018067302A2
WO2018067302A2 PCT/US2017/052318 US2017052318W WO2018067302A2 WO 2018067302 A2 WO2018067302 A2 WO 2018067302A2 US 2017052318 W US2017052318 W US 2017052318W WO 2018067302 A2 WO2018067302 A2 WO 2018067302A2
Authority
WO
WIPO (PCT)
Prior art keywords
liposomal particle
tlr
oligonucleotide
cells
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2017/052318
Other languages
English (en)
Other versions
WO2018067302A3 (fr
Inventor
Chad A. Mirkin
Jason A. WERTHEIM
Chenxia Monica GUAN
Lisa COLE
Jennifer Rachel FERRER
Gokay YAMANKURT
Shuya Wang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Northwestern University
Original Assignee
Northwestern University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Northwestern University filed Critical Northwestern University
Publication of WO2018067302A2 publication Critical patent/WO2018067302A2/fr
Publication of WO2018067302A3 publication Critical patent/WO2018067302A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00119—Melanoma antigens
    • A61K39/001192—Glycoprotein 100 [Gp100]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/04—Peptides being immobilised on, or in, an organic carrier entrapped within the carrier, e.g. gel, hollow fibre
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/812—Breast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present disclosure relates to liposomal particles, liposomal particles having encapsulated agents, methods of making the same, and uses thereof. Liposomal particles are useful in gene regulation and drug delivery.
  • SNA spherical nucleic acid
  • TLRs Toll-like receptors
  • Small unilamellar vesicles can be functionalized with polymers, peptides, DNA, and other molecules of interest by two distinct techniques.
  • a modified molecule of interest is added to the mixture of lipids, lipid film or hydration buffer during the synthesis of liposome. This approach results in a liposomes containing a functional molecule of interest on both inner and outer layers of the liposomal membrane.
  • structures created by this method are not stable at a size smaller than 80 nanometers (nm).
  • a SUV may be made by anchoring a substrate of interest into the lipid bilayer of the preformed vesicle (a "post modification technique").
  • This alternative approach yields a liposomal nanoparticle containing a functional molecule of interest on the outer layer of the liposomal membrane.
  • this alternative post-modification approach allows the creation of liposome of any size, even less than 50 nanometers.
  • Applications of the technology disclosed herein include pre-treatment of transplantable organs prior to transplantation surgery, modulation of ischemia reperfusion injury in a transplant setting, and attenuation of inflammation as a result of TLR activation (autoimmune disease, sepsis).
  • Advantages of the materials and methods disclosed herein include an increase in solubility of water insoluble small molecule drugs and biologies, and increase in circulation time/decrease in clearance rate of small molecules in vivo, and the ability to co-deliver multiple immunomodulatory molecules within a single entity.
  • Additional applications provided by the disclosure include the provision of selective and efficient TLR7/8 agonists; therapeutic cancer vaccines that can be tailored to a patient based on a tumor-associated antigen; and adjuvants of the immune system to be used on other types of vaccines.
  • Further advantages of the disclosure include but are not limited to the provision of an advantage over free RNA agonists: increased activation of TLR7/8 in a model cell line, and high cellular uptake into many cell types (including immune cells); and the provision of an advantage over chemical modified RNA agonists: increased potency of activating TLR7/8, and a single-entity construct that can deliver an antigen and activate TLRs within the same cell.
  • the disclosure provides a liposomal particle having a substantially spherical geometry and comprising a lipid bilayer comprising a plurality of lipid groups, a tumor antigen encapsulated therein, and an oligonucleotide on the surface of said liposomal particle.
  • the tumor antigen is a tumor lysate.
  • the oligonucleotide on the surface of said liposomal particle increases the activity of a toll-like receptor-9 (TLR-9).
  • a liposomal particle having a substantially spherical geometry and comprising a lipid bilayer comprising a plurality of lipid groups, a modulator of a first toll-like receptor (TLR) encapsulated in the liposomal particle, wherein said modulator is not an oligonucleotide, and an oligonucleotide on the surface of said liposomal particle that modulates the activity of a second toll-like receptor (TLR).
  • TLR toll-like receptor
  • the first TLR is TLR-4 and the second TLR is TLR-9.
  • the first TLR is TLR-9 and the second TLR is TLR-4.
  • the modulator of the first TLR is a small molecule.
  • the small molecule is TAK-242 (resatorvid), Oxidized 1 -palmitoyl-2-arachidonyl-sn- glycero-3-phosphorylcholine (OxPAPC), polymyxin B or eritoran.
  • the second TLR is TLR-9 and the oligonucleotide on the surface of said liposomal particle increases the activity of TLR-9.
  • the disclosure provides a liposomal particle having a substantially spherical geometry and comprising a lipid bilayer comprising a plurality of lipid groups, an oligonucleotide on the surface of said liposomal particle that increases the activity of a first tolllike receptor (TLR), and an oligonucleotide on the surface of said liposomal particle that increases the activity of a second toll-like receptor (TLR).
  • the first TLR is TLR-7 and the second TLR is TLR-8.
  • the oligonucleotide comprises or consists of the sequence as set out in SEQ ID NO: 10.
  • the oligonucleotide comprises or consists of the sequence as set out in SEQ ID NO: 1 1 .
  • the disclosure provides a liposomal particle having a
  • the modification is a phosphorothioate backbone or a phosphodiester backbone.
  • the oligonucleotide comprises a phosphodiester homopolymeric sequence. In some embodiments, the
  • homopolymer sequence comprises polyU.
  • the oligonucleotide comprises UCA or CpG.
  • the spacer is an internal spacer.
  • the lipid bilayer comprises 1 ,2- dioleoyl-sn-glycero-3 phosphatidylcholine (DOPC) and 5% 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-(6-azidohexanoyl) (DPPE-Azide).
  • DOPC dioleoyl-sn-glycero-3 phosphatidylcholine
  • DPPE-Azide 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-(6-azidohexanoyl)
  • DBCO dibenzocyclooctyl
  • said plurality of lipid groups comprises a lipid selected from the group consisting of the phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine families of lipids.
  • said lipid is selected from the group consisting of 1 ,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1 -palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1 ,2-distearoyl-sn-glycero-3-phospho-(1 '- rac-glycerol) (DSPG), 1 ,2-dioleoyl-sn-glycero-3-phospho-(1 '-rac-glycerol) (DOPG), 1 ,2- distearoyl-sn-glycero-3
  • the oligonucleotide is an oligonucleotide-lipid conjugate containing a lipophilic tethered group, wherein said lipophilic tethered group is adsorbed into the lipid bilayer.
  • the lipophilic tethered group comprises tocopherol or cholesterol.
  • tocopherol is chosen from the group consisting of a tocopherol derivative, alpha-tocopherol, beta-tocopherol, gamma-tocopherol and delta- tocopherol.
  • said oligonucleotide comprises RNA or DNA.
  • said RNA is a non-coding RNA.
  • said non-coding RNA is an inhibitory RNA (RNAi).
  • the RNAi is selected from the group consisting of a small inhibitory RNA (siRNA), a single- stranded RNA (ssRNA) that forms a triplex with double stranded DNA, and a ribozyme.
  • the RNA is a microRNA.
  • said DNA is antisense-DNA.
  • the disclosure also provides, in some embodiments, liposomal particles wherein diameter of said liposomal particle is less than or equal to about 50 nanometers.
  • the particle comprises from about 10 to about 80 oligonucleotides.
  • the particle comprises 70 oligonucleotides.
  • a liposomal particle of the disclosure comprises a modified oligonucleotide.
  • the disclosure provides a method of inhibiting expression of a gene comprising the step of hybridizing a polynucleotide encoding said gene product with one or more oligonucleotides complementary to all or a portion of said polynucleotide, said
  • oligonucleotide being attached to the liposomal particle of the disclosure, wherein hybridizing between said polynucleotide and said oligonucleotide occurs over a length of said
  • polynucleotide with a degree of complementarity sufficient to inhibit expression of said gene product.
  • expression of said gene product is inhibited in vivo.
  • expression of said gene product is inhibited in vitro.
  • said liposomal particle has a diameter about less than or equal to 50 nanometers.
  • said oligonucleotide comprises RNA or DNA.
  • said RNA is a non-coding RNA, and in some embodiments the non-coding RNA is an inhibitory RNA (RNAi).
  • the RNAi is selected from the group consisting of a small inhibitory RNA (siRNA), a single-stranded RNA (ssRNA) that forms a triplex with double stranded DNA, and a ribozyme.
  • siRNA small inhibitory RNA
  • ssRNA single-stranded RNA
  • ribozyme RNA
  • said RNA is a microRNA.
  • said DNA is antisense-DNA.
  • a method for modulating activity of at least two toll-like receptors comprising contacting a cell having the toll-like receptors with a liposomal particle of the disclosure.
  • the modulating is up-regulating TLR activity or down-regulating TLR activity.
  • the method is performed in vitro.
  • the method is performed in vivo.
  • the disclosure provides a method of treating or preventing ischemia reperfusion injury (IRI) in a patient, comprising administering a liposomal particle of the disclosure to the patient, thereby treating or preventing IRI.
  • IRI ischemia reperfusion injury
  • a liposomal particle having a substantially spherical geometry and comprising: a lipid bilayer comprising a plurality of lipid groups; an
  • oligonucleotide and an agent encapsulated in the liposomal particle, wherein the molar ratio of lipid to encapsulated agent is from about 5:1 to about 100:1 .
  • a liposomal particle said liposomal particle having a substantially spherical geometry and comprising: a lipid bilayer comprising a plurality of lipid groups; and an oligonucleotide comprising a modified backbone.
  • Figure 1 depicts TLR signaling.
  • Figure 2 provides a depiction of a LSNA containing TAK-242 and functionalized with a TLR9 antagonist oligonucleotide. The structure of TAK-242 is also shown.
  • Figure 3 is a schematic of LSNA synthesis using a DOPC small unilamellar vesicle and 3'-tocopherol conjugated DNA.
  • the final LSNA is approximately 60 nm in diameter.
  • Figure 4 shows TLR activity as measured in HEK-Blue reporter cells using the secreted alkaline phosphatase (SEAP) assay.
  • FIG. 5 depicts Liquid Chromatography Mass Spectrometry (LC-MS) analysis.
  • LC- MS was used to quantify TAK-242 encapsulated in liposomes.
  • the standard curve for 100:1 (lipid;TAK) liposome was made using a standard addition approach to minimize matrix effects.
  • Reserpine was the internal standard. Size and polydispersity indices were measured by DLS (Table 1 ).
  • Figure 6 shows the uptake of dye-labeled LSNAs in mouse macrophages.
  • Rhodamine-encapsulated liposomes were functionalized with Cy5-labeled DNA.
  • Raw 264.7 were incubated with the dual fluorophore-labeled LSNAs at 100nM DNA for 30 minutes.
  • Rhodamine channel shows the intracellular location of the liposome core (A), while Cy5 channel shows the intracellular location of the DNA (B).
  • Overlaid image of Rhodamine and Cy5 channels (C) shows a high degree of colocalization, indicating that LSNAs enters the cells as a single entity and do not degrade within the transfection time.
  • FIG. 7 shows TLR inhibition by free INH-18 or TAK-242 is specific.
  • RAW-Blue macrophages were stimulated with either 1826 CpG DNA (TLR9 ligand) or monophosphory lipid A (MPLA, TLR4 ligand).
  • TLR9 inhibition is clearly specific to INH-18 oligonucleotide (A), but TLR4 inhibition, though mostly inhibited by TAK-242, is slightly inhibited by INH-18 (B). Crosstalk between TLR4 and TLR9 pathways could explain this phenomenon.
  • FIG. 8 shows potent TLR4 and TLR9 inhibition by LSNAs in mouse and primary human cells.
  • Four different LSNAs were synthesized and RAW-Blue cells were treated with 500nM LSNAs to test for ability to modulate TLR activation (A-B).
  • Only particles containing INH-18 inhibitory sequences are capable of suppressing TLR9 activity (A) and more potently than free INH-18 DNA, while particles containing TAK-242 suppress TLR4 activity (B) comparable to free TAK-242.
  • CpG-induced human peripheral mononuclear blood cell (hPBMC) proliferation is inhibited in a dose dependent manner when treated with dual-targeting LSNAs (C).
  • Changing the DNA backbone of INH-18 from phosphodiester (PO) to phosphorothioate (PS) increases
  • Figure 9 provides a depiction of the TLR signaling pathway.
  • Figure 10 depicts functionalization of the LSNA surface with an inhibitory DNA sequence (INH-18).
  • FIG 11 show potent TLR4 and TLR9 inhibition by LSNAs in engineered TLR reporter cells.
  • Four different LSNAs were synthesized and HEK-Blue cells expressing mouse TLR4 or mouse TLR9 were treated with LSNAs (125nM by DNA, 75nM by TAK) to test for the ability to modulate TLR activation (A-B).
  • LSNAs 125nM by DNA, 75nM by TAK
  • Figure 12 demonstrates the kinetic advantage of using LSNAs.
  • the speed at which TLR9 activity is suppressed was assessed using HEK-Blue cells expressing mouse TLR9. Cells were treated for 4 hours simultaneously with agonist and antagonists (A) or with a 4 hour pulse of agonist followed by 8 hours with antagonist (B).
  • FIG. 13 depicts cytokine inhibition in TLR stimulated primary mouse splenocytes.
  • primary mouse splenocytes were isolated and treated with 500nM TLR9 agonist (A-E) or 500ng/ml TLR4 agonist (F) to stimulate cytokine production.
  • A-E 500nM TLR9 agonist
  • F 500ng/ml TLR4 agonist
  • Figure 15 shows results of a liver function test and IL-6 levels following IRI.
  • ALT alanine transaminase
  • ALP alkaline phosphatase
  • TBIL total bilirubin
  • CTL cholesterol
  • proinflammatory cytokine IL-6 proinflammatory cytokine IL-6.
  • Figure 16 depicts LSNA core synthesis.
  • Figure 17 provides final particle size and polydispersity index (PDI) of the liposomal cores as measured by DLS.
  • PDI polydispersity index
  • Figure 18 shows TLR9 suppression in HEK-Blue cells expressing mouse TLR9. Experiment was run overnight.
  • Figure 19 shows TLR4 suppression in HEK-Blue cells expressing mouse TLR4.
  • Figure 20 shows TLR9 suppression in HEK-Blue cells expressing mouse TLR9.
  • Figure 21 depicts an encapsulated spherical nucleic acid (SNA) vaccine, as well as the average size and average antigen loading per particle.
  • SNA spherical nucleic acid
  • Figure 22 shows a blue clear spot for SNA which is from the Cy5.
  • the graph shows the percentage of cells that were positive for Cy5 in the flow cytometer.
  • Figure 23 demonstrates that IS-SNAs co-deliver immunostimulatory oligonucleotides and tumor-associated antigen.
  • Y axis is the DNA and the X axis is the peptide. Each point is a cell and the four quadrants separate positive and negative cells.
  • Upper right corner is the percentage of cells positive for both peptide and DNA, indicating a doubling of the co-delivery efficiency with the SNA formulation compared to a simple mixture of oligonucleotide and peptide.
  • Figure 24 demonstrates that all of the cells were still being fully labeled, whereas on the right, multiple peaks to the right appear, indicating cells have divided. Each peak is one cell division so half of the cells have undergone multiple cell divisions with the peptide encapsulated SNA.
  • Figure 25 shows that antigen-specific Killer T-cell activity was induced by IS-SNAs in a tumor model.
  • Figure 26 shows the dose-dependent inhibition of CpG DNA-stimulated human PBMC proliferation by dual-targeting LSNAs measured by ( 3 H)-labeled thymidine incorporation.
  • Figure 27 shows data supporting the use of "top down” mass spectrometry analysis of intact proteins to identify unique protein signatures associated with IRI. The data show significant changes in proteoform expression between control and IRI-induced mouse kidneys.
  • Figure 28 shows LSNAs that exhibit enhanced activation of TLR7/8 over free RNA, and RNA transfected with a cationic transfection agent.
  • Figure 29 shows that the addition of LSNAs result in higher number of CD86+ receptors on dendritic cells than when they are introduced to RNA instead. These results are comparable to Resiquimod, a TLR7/8 agonist that was used as a positive control.
  • Figure 30 shows that LSNAs induce the production of IL-6 (released in late endosomes) more so than free RNA, a control SNA, or untreated.
  • Figure 31 is a schematic of the process for encapsulating proteins or peptides in a spherical nucleic acid ⁇ e.g., a LSNA).
  • Figure 32 shows that greater than 94% of the DOPC lipid is incorporated into liposomes as measured using a phosphotidyl choline assay kit.
  • Figure 33 shows that encapsulation efficiencies for peptides was 1 1 -17% of the starting material, and for labeled ovalbumin proteins, it was 29-31 %. It was also observed that the encapsulation efficiency was independent of the starting concentration of the peptide or protein.
  • Figure 34 shows (A) Scheme of LSNA functionalization: DOPC core functionalized with cholesterol-modified RNAs. (B) DLS of bare liposomes compared to LSNAs (in nm). (C) Gel electrophoresis of Cy3.5 labeled LSNAs compared to free 5'cholesterol ssRNA show an increase in size as determined by reduction in mobility.
  • IS-r-LSNAs immunostimulatory RNA-based LSNAs
  • Figure 36 shows (A) Confocal image shows high cellular uptake of Cy3.5 labeled SNAs into murine bone marrow derived dendritic cells (BMDCs) compared to free ssRNA or transfected ssRNA at 24 hours. Red denotes Cy3.5 labeled ssRNA and DAPI denotes a nuclear stain; scale bar indicates 5um.
  • BMDCs murine bone marrow derived dendritic cells
  • Figure 37 shows Z-stack of confocal images from Figure 36A were taken at 0.5 urn slices; scale bar on right bottom indicates 5 urn.
  • IS-r-LSNAs upregulate co-stimulatory receptor CD86 and MHCII in antigen presenting cells (pDC: PDCA + , CD1 1 c med , CD19 " , B cells: CD19 + , B220 + , and macrophages: F4/80 + ) to a higher extent than free or cationically transfected ssRNA after a 24h incubation.
  • pDC PDCA + , CD1 1 c med , CD19 "
  • B cells CD19 + , B220 + , and macrophages: F4/80 +
  • NK NK1 . ⁇ , CD49b + , CD 3 "
  • T cells CD3 + ).
  • Figure 39 shows (A) IS-r-LSNAs enhance antigen presentation rates on pDCs (PDCA + , CD1 1 c med , CD19 " ), B cells (CD19 + , B220 + ), and macrophages (F4/80 + ), compared to that of free or cationically transfected ssRNA, after a 24 hour incubation, as quantified using flow cytometry.
  • PDCA + , CD1 1 c med , CD19 " ), B cells (CD19 + , B220 + ), and macrophages (F4/80 + ) compared to that of free or cationically transfected ssRNA, after a 24 hour incubation, as quantified using flow cytometry.
  • IS-r-LSNAs also increased antigen presentation rates of
  • (B) IS-r-LSNA primed DCs (24 hours) induce greater OT-I CD8+ T cell proliferation than that of free or cationically transfected RNA after 72 hours. In addition, LSNA induce similar CD8+ T cell proliferation as resiquimod. The results are representative results from three independent experiments; bars indicate mean ⁇ SD (n 3). ** : p ⁇ 0.01 between LSNA DR and all groups except resiquimod, where there is no statistical significance between the two groups.
  • Figure 40 shows that Bare DOPC SUVs have a smooth shell (left), unlike a functionalized IS-r-LSNA, which has a more "bumpy" surface due to the presence of RNA (right two). 5' cholesterol modified ssRNA can self-assemble or aggregate to form structures that make them visible under cryoEM (second from left). The clustering of RNA does not appear in the background in the LSNA sample, and instead, can be seen on the outside of the liposomal shell, confirming the successful functionalization or RNA onto liposomes. All scale bars are at 50nm.
  • Figure 41 shows results of experiments probing structure/function relationships between TLRs7/8 and IS-r-LSNAs.
  • Figure 43 shows that overnight incubation of Hek-Blue cells with chloroquine does not decrease cell viability.
  • Figure 45 shows results of experiments to determine whether IS-r-LSNAs can specifically maturate DCs, all constructs were incubated with positively selected (CD1 1 c+ ) DCs for 24h, and analyzed using flow cytometry to look at the DC maturation through the
  • Figure 46 shows a comparison between ssRNA-DR as functionalized onto r-LSNAs or cationically transfected for cytokine secretion (A) and primary immune cell activation (B).
  • Figure 47 depicts an immune-stimulating spherical nucleic acid vaccine as a treatment for triple negative breast cancer.
  • Figure 48 is a schematic showing how SNAs are internalized by specialized immune cells, causing these cells to turn on the immune system. The activated cells then interact and communicate with T-cells, enabling the T-cells to recognize and kill tumor cells that express the antigens.
  • Figure 49 shows that f luorescently labeled SNAs are rapidly internalized by macrophages (nucleus stained).
  • Figure 50 shows that SNA vaccine treatment reduced the number of lung metastases in 4T1 tumor-bearing mice compared with untreated mice. Twenty-one days after treatment mouse lungs were cultured ex vivo, the 4T1 cells residing in these organs were stained (blue) and counted to quantify the number of metastases. One dot represents one metastasis.
  • Figure 51 is a SDS-PAGE gel showing the generation of TNBC lysates through five free-thaw cycles of 4T1 cells.
  • the SDS-Page gel shows the proteins in the lysates span molecular weights of 10-260 kDa. This demonstrates that the proteins loaded into the liposomes are a mixture of different proteins.
  • Figure 52 depicts a schematic of how the lysate loaded liposomal SNAs were synthesized.
  • Figure 53 demonstrates the high cellular uptake of TNBC lysate or CpG DNA into antigen presenting cells, including macrophages and dendritic cells, after one hour incubation time in vitro.
  • Top/Bottom Left Encapsulation of protein lysates into LSNAs enables higher cellular uptake of lysates into macrophages and dendritic cells compared with lysates encapsulated within bare liposome as demonstrated by confocal microscopy (brighter green signal inside cells) and flow cytometry (higher fluorescent intensity).
  • Top/Bottom Right Encapsulation of protein lysates into LSNAs enables higher cellular uptake of lysates into macrophages and dendritic cells compared with lysates encapsulated within bare liposome as demonstrated by confocal microscopy (brighter green signal inside cells) and flow cytometry (higher fluorescent intensity).
  • Top/Bottom Right Encapsulation of protein lysates into
  • Arranging DNA into a spherical architecture leads to greater cellular uptake compared with free DNA.
  • Figure 54 demonstrates that co-delivery of TNBC antigen and CpG DNA to the same cell is enhanced when formulated as an LSNA. Co-delivery of lysate and DNA into the same cell is increased when formulated into a SNA.
  • Cells were treated with three different combinations of samples: 1 ) Cy5-CpG SNA and AlexaFluor 488 (AF488)-labeled lysate encapsulated within bare liposomes, 2) free Cy5-labeled DNA and AF488-labeled lysate encapsulated within SNA, and 3) Dual labeled SNAs.
  • FIG 55 provides a schematic showing the activation of dendritic cells (DCs).
  • FIG 56 shows that TNBC LSNAs activate antigen presenting cells.
  • Protein lysate LSNAs can activate mouse dendritic cells through toll-like receptor 9 signaling from the DNA, and also through delivery of the lysate.
  • Protein lysates are known to active dendritic cells through proteins that are released during necrotic cell death that are meant to signal to surrounding cells that there is danger in the area that caused this type of death.
  • Activated dendritic cells increase their expression of co-stimulatory molecules (CD80, CD40, CD86, MHCII) and secretion of pro-inflammatory cytokines (TNFa, IL-12, IL-1 ⁇ , IL-6).
  • Control - GpC DNA SNA non-Toll-like receptor 9 activating DNA
  • TLR9 - CpG DNA SNA Toll-like receptor 9 activating DNA
  • Empty - nothing inside the liposome Lysate - liposomes with lysates encapsulated inside.
  • Figure 57 is a schematic showing the vaccine protocol studies as conducted in mice. The figure also shows results of tumor growth monitoring in the mice. The tumor volume of 4T1 tumors in the right mammary gland measured overtime. Control LSNAs are liposomes encapsulating lysates and GpC DNA. TLR9 LSNAs are liposomes encapsulating lysates and CpG DNA. Treatment with TLR9 LSNAs has a small effect on tumor volume; slowing growth at day 20 compared with untreated and control LSNA groups.
  • Figure 58 shows that the percent of CD8+ T-cells within the tumor increased with TNBC LSNA treatment.
  • Figure 59 shows that the percent of myeloid derived suppressive cells within the tumor decreased with TNBC LSNA treatment.
  • Figure 60 provides data demonstrating that the TNBC LSNA vaccine reduced lung metastasis in a mouse model. Each dot represents one metastasis. * p ⁇ 0.05. The number of lung metastasis on day 21 was greatly reduced after TLR9 LSNA treatment compared with untreated or control LSNA groups. This was measured by counting the number of 4T1 cells that reached the lungs after implantation into the right mammary gland. Fewer 4T1 cells were able to colonize in the lungs in mice treated with TLR9 SNAs.
  • FIG. 61 shows the physical characterization of liposomal nanoparticles.
  • Protein encapsulation was measured using bicinchoninic acid (BCA) assay DNA concentration measured using ultra-violet visible spectroscopy (A 26 o)- It was determined that there was 1 .1 (0.65) mg protein per ⁇ DNA (mean ( ⁇ standard deviation) of 3 batches).
  • CryoEM images show the SNAs are spherical and approximately 50-100 nm in diameter after encapsulating a mixture of proteins.
  • Agarose gel electrophoresis and dynamic light scattering both indicate an increase in size after functionalization with DNA. In the agarose gel, the SNA doesn't migrate as far in the gel due to its larger size compared with free DNA.
  • the hydrodynamic diameter of bare liposomes is 73.4 nm and SNAs is 87.7 nm suggesting successful functionalization of DNA.
  • Figure 62 demonstrates that IS-SNAs co-deliver immunostimulatory oligonucleotides and tumor-associated antigen.
  • Spherical Nucleic Acids are a class of nanoparticles functionalized with a dense layer of oligonucleotides surrounding an exchangeable core. This nucleic acid shell imparts several functionalities: the oligonucleotide coating forms a highly concentrated salt cloud that decreases endonuclease activity on the nanoparticle surface, and interacts with cell surface proteins, resulting in high cellular uptake in virtually all cell lines. The combination of these unique characteristics allows SNAs to behave as easily tailorable, single-entity agents that serve a variety of applications, such as cancer therapeutics.
  • Liposomes are spherical, self-closed structures in a varying size range consisting of one or several hydrophobic lipid bilayers with a hydrophilic core.
  • the diameter of these lipid based carriers range from 0.15-1 micrometers, which is significantly higher than an effective therapeutic range of 20-100 nanometers.
  • SUVs small unilamellar vesicles
  • the disclosure provides RNA based immunostimulatory SNAs (IS- SNAs). These IS-SNAs are shown herein to exhibit over 30-fold increase of secreted NF- ⁇ in murine macrophages compared to the free strand delivered using a cationic transfection agent (no activation was seen with just the free strand).
  • IS- SNAs RNA based immunostimulatory SNAs
  • treatment of mouse dendritic cells with RNA-based IS-SNAs in the presence of model tumor antigen improves the rate of antigen presentation, one important step in immunizing against cancer. This observation sets the foundation for the development of RNA-SNA based antitumor vaccines.
  • a description of an embodiment of the disclosure in terms of a small unilamellar vesicle (SUV), a liposomal SNA (LSNA), IS-SNA, IS-r-LSNA, a liposomal particle, or a spherical nucleic acid (SNA) may also be applicable to an embodiment that uses any of the other foregoing terms.
  • a method of regulating gene expression using a liposomal SNA may also be described herein as a method of regulating gene expression using a liposomal particle.
  • SUVs Small unilamellar vesicles
  • LSNAs Small unilamellar vesicles
  • a range includes each individual member.
  • a group having 1 -3 members refers to groups having 1 , 2, or 3 members.
  • a group having 6 members refers to groups having 1 , 2, 3, 4, or 6 members, and so forth.
  • the modal verb "may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb "may” refers to an affirmative act regarding how to make or use an aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb "may” has the same meaning and connotation as the auxiliary verb "can.”
  • the articles “a” and “an” refer to one or to more than one (for example, to at least one) of the grammatical object of the article.
  • “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20-25 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.
  • the lUPAC nomenclature can be derived with chemical structure drawing software programs, such as ChemDraw® (PerkinElmer, Inc.), ChemDoodle® (iChemLabs, LLC) and Marvin (ChemAxon Ltd.).
  • ChemDraw® PerkinElmer, Inc.
  • ChemDoodle® iChemLabs, LLC
  • Marvin Marvin
  • the present disclosure describes novel particles, termed liposomal particles, methods of making the same, and uses of these particles.
  • the present liposomal particles are advantageous over other known liposomal based materials in that they are stable at a particle size that is smaller than other known liposomal particles, and the dense layer of DNA increases particle stability in a body, and therefore increases the circulation rate of liposomal vesicles, which improves bio-distribution of these particles inside the body.
  • RNA based structures hold promise in increased stability, immune cell uptake, and potency of TLR7/8 activation of RNA when compared to free or cationically transfected sequences.
  • These immunostimulatory RNA-based LSNAs are also modular in design, allowing for the encapsulation of peptide antigens that can stimulate an antigen-specific response in CD8+ T cells. Further, these modular IS-r-LSNAs activate both arms of the immune system, which is useful for many applications including cancer immunology, autoimmune diseases or tolerance.
  • Spherical nucleic acids a class of nanoparticles composed of interchangeable cores functionalized with a dense oligonucleotide shell, can alter the way a cell sees nucleic acids [Cutler et al., J Am Chem Soc 2012, 134 (3), 1376-91 ; Mirkin et al., Nature 1996, 382 (6592), 607-9.].
  • nucleic acids On their own, nucleic acids have low cellular uptake in cells due to their negative charge; SNAs, due to their highly oriented shell of oligonucleotides, have increased cell surface receptor interactions, resulting in high cellular uptake in virtually all cells, including immune cells [Rosi et al., Science 2006, 312 (5776), 1027-30; Giljohann et al., Nano Lett 2007, 7 (12), 3818-21 ].
  • the salt cloud surrounding SNAs reduces endonuclease activity on the nanoparticle surface, minimizing the concern of RNA instability [Seferos et al., Nano Lett 2009, 9 (1 ), 308-1 1 ; Patel et al., Mol Pharm 201 1 , 8 (4), 1285-91 .].
  • SNAs are also non-cytotoxic [Massich et al., ACS Nano 2010, 4(10), 5641 -6; Massich et al., Mol Pharm 2009, 6 (6), 1934- 40], unless they are designed specifically to activate the immune system, allowing them to be used for a variety of biological applications.
  • TLRs are a class of pathogen recognition receptors (PRRs) that that play an important role in our innate immune system by recognizing pathogen associated membrane patterns (PAMPs) [O'Neill et al., Nat Rev Immunol 2013, 13 (6), 453-60; Blasius et al., Immunity 2010, 32 (3), 305- 15; Kawai et al., Nat Immunol 2010, 1 1 (5), 373-84; Rakoff-Nahoum et al., Nat Rev Cancer 2009, 9 (1 ), 57-63].
  • PRRs pathogen recognition receptors
  • PAMPs pathogen associated membrane patterns
  • TLRs reside mostly in immune cells, and upon PAMP binding, initiate an innate immunity response resulting in the activation of many signaling transduction pathways relevant in inflammation, cytokine release, and adaptive immunity [Blasius et al., Immunity 2010, 32 (3), 305-15; Kawai et al., Nat Immunol 2010, 1 1 (5), 373-84; Rakoff-Nahoum et al., Nat Rev Cancer 2009, 9 (1 ), 57-63].
  • the use of TLR ligands to activate immune cells is advantageous over other types of adjuvants due to their well-characterized molecular structure, ligand-receptor specificity, and signaling pathways in a whole host of immune cells.
  • TLR9 endosomal TLRs, which that recognize nucleic acids of bacterial (TLR9) and viral (TLR7/8 and TLR3) origin [Blasius et al., Immunity 2010, 32 (3), 305-15; Junt et al., Nat Rev Immunol 2015, 15 (9), 529-44; Schon et al., Oncogene 2008, 27 (2), 190-9].
  • TLRs in many immune cells justifies the increased interest in the development of efficient ligands for many therapeutic applications, such as cancer and autoimmune disorders ⁇ i.e., rheumatoid arthritis, and systemic lupus erythematosus) [O'Neill et al., Pharmacol Rev 2009, 61 (2), 177-97].
  • the activation of these TLRs results in a rapid and potent dose of type I IFNs and pro-inflammatory cytokines, resulting in both innate and adaptive immunity [Reizis et al., Annual review of immunology 201 1 , 29, 163-83].
  • RNA oligomers heavily relies on the use of cytotoxic transfection reagents [Bourquin et al., J Immunother 2010, 33 (9), 935-44; Nguyen et al., Proc Natl Acad Sci USA 2012, 109 (14), E797- 803].
  • SNAs due to their unique properties imparted by the oligonucleotide shell, can overcome instability and low uptake challenges faced by oligonucleotides.
  • IS-r-LSNAs ssRNA TLR7/8 agonist functionalized liposomal SNAs
  • RNA oligonucleotides can be designed to activate toll-like receptors (TLRs) 7/8.
  • TLRs toll-like receptors
  • orienting these RNA agonists in a SNA form can significantly increase their efficacy. This increase in activity can be attributed to the SNAs' rapid and high cellular uptake and their trafficking mechanism through the endocytic pathway.
  • These potent TLR agonists are used to develop a therapeutic cancer vaccine that specifically induces cancer cell death through the co-delivery of an antigen delivered using SNA composed of a liposomal core (synthesized using FDA approved materials).
  • Immuno-modulatory spherical nucleic acids have shown great promise in activating and suppressing toll-like receptor (TLR) 9 for applications in anti-tumor immunity and autoimmune diseases.
  • TLR toll-like receptor
  • APCs antigen-presenting cells
  • TLR 7/8 agonists ⁇ e.g., resiquimod
  • RNA-based TLR 7/8 agonists while specific, can be easily degraded by nucleases and lack an efficient means of cellular uptake.
  • This disclosure includes use of liposomal SNAs to activate TLR 3, 7 and 8 via RNA immuno-stimulatory oligonucleotides.
  • SNAs Due to their dense oligonucleotide shell, SNAs exhibit increased interactions with cell surface proteins resulting in high cellular uptake in virtually all cell lines while localizing mainly in endosomal compartments, decreased activity of endonucleases on the nanoparticle surface due to a high local salt concentration, and also no cytotoxicity. As a result, SNAs are perfectly suited to act as single-entity RNA agonists for activating APCs.
  • TLR7/8 RNA agonists have been developed in the past, many suffer from stability and delivery challenges.
  • This disclosure describes how insertion of single stranded TLR7/8 RNA agonists into a liposomal core to form liposomal spherical nucleic acids (LSNAs) offers improved transfection, internalization, and activation of TLR 7/8.
  • LSNAs liposomal spherical nucleic acids
  • the 3D radial orientation of the nucleic acids increase the interaction of nucleic acids on LSNAs to cell surface proteins to yield higher uptake, while the high salt cloud on the LSNA offers stability against endonucleases.
  • LSNAs activate TLR7/8 in a model cell line (human embryonic kidney 293 reporter cells expressing TLR7/8, Hek-Blue) with higher potency than free RNA, or RNA transfected with a cationic transfection agent, likely due to the multivalent binding of RNA to TLR7/8 in endosomal compartments.
  • This increase in potency also translates into increased activation of dendritic cells, through the upregulation of co-stimulatory markers and increase in cytokine secretion, of LSNAs over their free RNA counterparts.
  • the present disclosure provides for the use of dual-targeting LSNAs for attenuating inflammation.
  • Toll-like receptors TLRs
  • TLRs Toll-like receptors
  • IRI ischemia reperfusion injury
  • LSNAs dual TLR-inhibiting liposomal spherical nucleic acids
  • LSNAs are biodegradable nanoparticles synthesized from non-toxic, FDA-approved materials.
  • the modularity of the LSNA construct permits encapsulation and surface functionalization of a number of small molecule drugs and biologically active molecules for tailoring the construct to a wide range of potential biomedical applications. This, combined with its chemical features and biological properties, such as rapid and efficient cellular uptake, make LSNAs a strong candidate for inhibiting TLR activation in vivo.
  • the LSNAs of the disclosure are designed to inhibit TLR4 and TLR9, which have both been implicated in inflammatory diseases. It is shown herein that, despite differences in the cellular location of TLR4 and TLR9, LSNAs are capable of suppressing the activity of both targets.
  • the disclosure also provides, in some aspects, methods of reducing inflammation in a patient in need thereof, the method comprising the step of administering a LSNA of the disclosure to the patient in an amount sufficient to reduce inflammation.
  • LSNA inhibit TLR-4 and/or TLR-9.
  • an "inflammatory condition” means a disease, disorder or condition characterized by inflammation of body tissue or having an inflammatory component. These include local inflammatory responses and systemic inflammation. Examples of such inflammatory conditions include: transplant rejection, including skin graft rejection;
  • osteoarthritis and bone diseases associated with increased bone resorption inflammatory bowel diseases such as ileitis, ulcerative colitis, Barrett's syndrome, and Crohn's disease;
  • inflammatory lung disorders such as asthma, adult respiratory distress syndrome, and chronic obstructive airway disease; inflammatory disorders of the eye including corneal dystrophy, trachoma, onchocerciasis, uveitis, sympathetic ophthalmitis and endophthalmitis; chronic inflammatory conditions of the gums, including gingivitis and periodontitis; tuberculosis; leprosy; inflammatory diseases of the kidney including uremic complications, glomerulonephritis and nephrosis; inflammatory conditions of the skin including sclerodermatitis, psoriasis and eczema; inflammatory diseases of the central nervous system, including chronic demyelinating diseases of the nervous system, multiple sclerosis, AIDS-related neurodegeneration and Alzheimer's disease, infectious meningitis, encephalomyelitis, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and viral or autoimmune encephalitis; autoimmune disorders, immune-complex vas
  • a systemic inflammation of the body exemplified by gram- positive or gram negative shock, hemorrhagic or anaphylactic shock, or shock induced by cancer chemotherapy in response to pro-inflammatory cytokines, e.g., shock associated with pro-inflammatory cytokines.
  • shock can be induced, e.g., by a chemotherapeutic agent used in cancer chemotherapy.
  • Treatment of an inflammatory condition refers to administering a compound or a composition of the invention to a subject, who has an inflammatory condition, a symptom of such a condition or a predisposition towards such a condition, with the purpose to cure, relieve, alter, affect, or prevent the inflammatory condition, the symptom of it, or the predisposition towards it.
  • TLRs Toll-like receptors
  • Figure 2 Schott al., Nature biotechnology. 2007;25(8):825-6
  • DAMPs damage-associated molecular patterns
  • TLR signaling plays a central role in the initiation and propagation of the proinflammatory response, ultimately converging upon NF- ⁇ and resulting in the production of cytokines and chemokines, immune cell activation, migration and proliferation, and the consequent shaping of the adaptive immune response.
  • TLR1 -9 Persistent over-stimulation of TLRs leads to the development and progression of several diseases, such as bacterial and viral infections and sterile inflammatory disorders. TLR1 -9 is highly conserved across humans and rodents. This high degree of conservation across species makes these receptors attractive therapeutic targets.
  • Ischemia occurs when oxygen is cut off to an organ and the subsequent reperfusion injury initiates an inflammatory cascade upon reestablishment of blood flow.
  • IRI damages the organ's cellular parenchyma, releasing DAMPS that initiate the inflammatory cascade via TLR signaling and further compromise organ function due to resulting inflammation.
  • static, cold preservation e.g. University of Wisconsin solution
  • DAMPS that initiate the inflammatory cascade via TLR signaling and further compromise organ function due to resulting inflammation.
  • the ischemia-reperfusion injury can be associated with ischemic and post-ischemic events in organs and tissues, including but not limited to thrombotic stroke; myocardial infarction;
  • angina pectoris embolic vascular occlusions; peripheral vascular insufficiency; splanchnic artery occlusion; arterial occlusion by thrombi or embolisms, arterial occlusion by non-occlusive processes such as following low mesenteric flow or sepsis; mesenteric arterial occlusion;
  • ischemic acute renal failure ischemia-reperfusion injury to the cerebral tissue; intestinal intussusception; hemodynamic shock; tissue dysfunction; organ failure; restenosis;
  • the ischemia-reperfusion injury can be alternatively following conditions including but not limited to procedures such as cardiac surgery; organ surgery; organ transplantation; angiography; cardiopulmonary and cerebral resuscitation.
  • TLR4 located at the cell surface, is a central mediator of IRI following activation by endogenous, damage-associated, non-pathogenic ligands (Altemeier et al., PloS one. 2013;8(10):e77123; Chang et al., Journal of investigative surgery : the official journal of the Academy of Surgical Research.
  • TLR4 signals through two pathways, one dependent upon MyD88 and the other independent of this adapter protein ( Figure 1 ).
  • TLR4 Activation of the TLR4 signal cascade culminates in NF- ⁇ and IFN3 activation and production of pro-inflammatory cytokines and chemokines, such as TNF-a, IL-6, IL-10, IFNy and IL-1 ⁇ .
  • TLR4 is expressed in nearly all cells of the body.
  • TLR9 is located in the endosome and recognizes unmethylated bacterial cytosine- phosphate-guanine (CpG) DNA, denatured host DNA, and a number of synthetic oligodeoxynucleotides that inhibit or activate TLR9 signaling.
  • CpG cytosine- phosphate-guanine
  • the ability of TLR9 to recognize endogenous DAMPs released by injured, apoptotic and necrotic cells indicates an involvement in the pro-inflammatory response associated with IRI (Chang et al., Journal of investigative surgery : the official journal of the Academy of Surgical Research. 2012;25(4):271 - 7; Bamboat et al., Hepatology. 2010;51 (2):621 -32; Huang et al., Hepatology. 2015;62(2):600- 14).
  • TLR9 is expressed in many cells that express TLR4, including monocytes and dendritic cells.
  • TLR9 One approach to down-regulating overactivated TLRs is to competitively inhibit the receptor with soluble factors such as small molecules or antibodies. This approach is useful for targeting cell surface TLRs but is not effective against intracellular TLRs, such as TLR9.
  • TLR-inhibitors leads to their rapid clearance by the renal and reticuloendothelial system.
  • Another approach to inhibit TLRs is through oligonucleotides, such as small interfering RNA (siRNA) or inhibitory DNA or RNA that bind endosomal TLRs.
  • siRNA small interfering RNA
  • oligonucleotide-based approaches conventional delivery is impeded by the negative charge on oligonucleotides and requires cytotoxic transfection reagents.
  • LSNAs Liposomal spherical nucleic acids
  • LSNAs bound to its surface ( Figure 1 ) (Banga et al., Journal of the American Chemical Society. 2014;136(28):9866-9).
  • the chemistry and geometry of LSNAs confers unique properties that are distinct from either the core or oligonucleotide ligands alone. Relative to free oligonucleotides, LSNAs have: 1 ) increased protection against nuclease degradation; 2) higher binding affinity to complimentary oligonucleotides; 3) high cellular uptake without the use ancillary transfection reagents; and 4) are non-immunogenic (Cutler et al., Journal of the American Chemical Society.
  • LSNAs are also highly modular; the core and the oligonucleotides are easily altered to carry fluorophores for imaging and diagnostic purposes, small molecules, peptides or proteins for therapeutic and/or targeting purposes, and the oligonucleotides themselves can be modified to function as antisense DNA, small interfering RNA (siRNA), or microRNAs for gene regulation (Giljohann et al., Journal of the American Chemical Society. 2009;131 (6):2072-3; Halo et al., Proceedings of the National Academy of Sciences of the United States of America. 2014;1 1 1 (48):17104-9; Mirkin et al., Oncotarget.
  • siRNA small interfering RNA
  • the disclosure also provides, in some aspects, LSNAs and methods for treating or preventing IRI, the method comprising the step of administering a LSNA of the disclosure that inhibits TLR-4 and TLR-9.
  • the disclosure also provides, in various aspects, a spherical nucleic acid (SNA) vaccine designed to treat cancer.
  • the cancer is triple negative breast cancer (TNBC) tumors.
  • TNBC triple negative breast cancer
  • a SNA vaccine of the disclosure designed against TNBC reduces the overall size of the primary tumor and drastically reduces the number of lung metastases.
  • SNAs are a new class of molecules that can modulate the immune system through specific interactions with immune cells.
  • Immune-stimulatory SNAs consist of a dense shell of nucleic acids that is radially oriented and attached to a liposomal nanoparticle core (Figure 47) [Banga et al., J. Am. Chem. Soc. 136, 9866-9869 (2014); Radovic-Moreno et al., Proc. Nat. Acad. Sci. USA. 1 12, 3892-3897 (2015)].
  • the nucleic acids are specifically designed to have multiple cytosine (C) and guanine (G) nucleotide repeats (CpG) within the linear sequence.
  • TNBC toll-like receptor 9
  • Immunotherapy is a potential treatment for TNBC because the number of tumor-infiltrating lymphocytes has been correlated to increased overall survival.
  • certain aspects of the disclosure are directed to providing spherical nucleic acid vaccines (SNAs) for TNBC by delivering toll-like receptor 9 agonist DNA and TNBC-specific antigens to antigen presenting cells in vivo, and elicit an anti-tumor immune response against established TNBC tumors and metastasis.
  • SNAs spherical nucleic acid vaccines
  • Protein cell lysates are being used as an antigen source for a LSNA cancer vaccine against triple negative breast cancer (TNBC) because TNBC does not have any known antigens or neoantigens due to a lack of molecular similarity between TNBC tumors. Therefore, whole cell lysates are being used as a large mixture of antigens that can direct an immune response.
  • the liposomal particle comprises tumor lysate.
  • Tumor lysis can be effected using any of a number methods, such as repeated freezing and thawing of the tumor; physical disruption by homogenization or pulverization; or by chemically induction by, e.g., exposure to a hyper- or hypotonic solution or one or more non-ionic detergents.
  • the tumor lysate is obtained from a tumor associated with, e.g., breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, liver cancer, pancreatic cancer, cervical cancer, ovarian cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, and brain cancer.
  • a tumor associated with e.g., breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, liver cancer, pancreatic cancer, cervical cancer, ovarian cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, and brain cancer.
  • the liposomal core of the immune-stimulatory SNA is important as the core is used to encapsulate TNBC-specific antigens, which are a mixture of proteins expressed by TNBC cells and captured after cellular lysis.
  • TNBC-specific antigens which are a mixture of proteins expressed by TNBC cells and captured after cellular lysis.
  • the SNA architecture enables co-delivery of the encapsulated antigens and CpG DNA to the same immune cells.
  • the binding of the CpG-rich SNA to TLR9 leads to cellular activation and the immune system being turned on against the antigens that were delivered such that cytotoxic T-cells are primed to kill only the cells that express TNBC associated antigens (Figure 48).
  • SNAs are rapidly internalized into immune cells (Figure 49), with 100% of the SNAs being internalized after only two hours.
  • the SNA architecture increased the co-delivery of DNA and antigens into the same cell by 50% compared with delivering the DNA and antigens as free entities.
  • the delivered DNA activates immune cells through TLR9, resulting in increased secretion of pro-inflammatory cytokines, which are important biomolecules for driving a long- lasting immune response in vivo.
  • the present disclosure provides liposomal particles for use in methods including but not limited to regulation of gene expression and activation of TLRs.
  • the liposomal particle has at least a substantially spherical geometry, an internal side and an external side, and comprises a lipid bilayer.
  • the lipid bilayer is comprised of a first-lipid and a second-lipid.
  • the first-lipid and second-lipid are, in some embodiments, the same. In further embodiments, the first-lipid and second-lipid are different.
  • the first-lipid is chosen from the phosphocholine family of lipids or the
  • the first-lipid is chosen from group consisting of 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dimyristoyl-sn- phosphatidylcholine (DMPC), 1 -palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1 ,2- distearoyl-sn-glycero-3-phospho-(1 '-rac-glycerol) (DSPG), 1 ,2-dioleoyl-sn-glycero-3-phospho- (1 '-rac-glycerol) (DOPG), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1 ,2-di-(9Z-o)
  • DOPC 1,2-dioleoyl-sn
  • the second-lipid is chosen from the phosphocholine family of lipids or the
  • the second-lipid is chosen from group consisting of 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dimyristoyl-sn- phosphatidylcholine (DMPC), 1 -palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1 ,2- distearoyl-sn-glycero-3-phospho-(1 '-rac-glycerol) (DSPG), 1 ,2-dioleoyl-sn-glycero-3-phospho- (1 '-rac-glycerol) (DOPG), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1 ,2-di-(9Z-o
  • DOPC 1 ,2-di-(9Z-o
  • the liposomal particle further comprises a tocopherol modified oligonucleotide wherein the lipophilic end of the tocopherol modified oligonucleotide is absorbed into the lipid bilayer.
  • the tocopherol is chosen from the group consisting of alpha-tocopherol, beta-tocopherol, gamma-tocopherol and delta-tocopherol.
  • the non-lipophilic end of the tocopherol modified oligonucleotide is an oligonucleotide. This oligonucleotide is, in various embodiments, either RNA or DNA.
  • the RNA can be an inhibitory RNA (RNAi) that performs a regulatory function, and in various embodiments is selected from the group consisting of a small inhibitory RNA (siRNA), an RNA that forms a triplex with double stranded DNA, and a ribozyme.
  • RNAi small inhibitory RNA
  • the RNA is microRNA that performs a regulatory function.
  • the DNA is optionally an antisense-DNA.
  • the RNA is a piwi-interacting RNA (piRNA).
  • the disclosure provides a liposomal particle, said liposomal particle having a substantially spherical geometry, said liposomal particle comprising a lipid bilayer comprising a plurality of lipid groups; and an oligonucleotide.
  • the oligonucleotide is a modified oligonucleotide.
  • the plurality of lipid groups comprises a lipid selected from the group consisting of the phosphatidylcholine,
  • the oligonucleotide in further embodiments is an oligonucleotide-lipid conjugate containing a lipophilic tethered group, wherein said lipophilic tethered group is adsorbed into the lipid bilayer.
  • the lipophilic tethered group comprises, in various embodiments, tocopherol, palmitoyl, dipalmitoyl, stearyl, distearyl, or cholesterol.
  • a liposomal particle as described herein comprises from about 1 to about 400 oligonucleotides on its surface.
  • a liposomal particle comprises from about 10 to about 100, or from 10 to about 90, or from about 10 to about 80, or from about 10 to about 70, or from about 10 to about 60, or from about 10 to about 50, or from about 10 to about 40, or from about 10 to about 30, or from about 10 to about 20, or from about 50 to about 100, or from about 60 to about 100, or from about 70 to about 100, or from about 80 to about 100, or from about 90 to about 100 oligonucleotides on its surface.
  • a liposomal particle comprises at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, or 400 oligonucleotides on its surface.
  • an agent is encapsulated within a LSNA.
  • a tumor antigen is encapsulated in a LSNA.
  • a modulator of a first TLR is encapsulated in a LSNA.
  • a small molecule is encapsulated in a LSNA as disclosed herein.
  • the term "small molecule,” as used herein, refers to a chemical compound, for instance a chemotherapeutic or peptidometic that may optionally be derivatized, or any other low molecular weight organic compound, either natural or synthetic.
  • low molecular weight is meant compounds having a molecular weight of less than 1000 Daltons, typically between 300 and 700 Daltons.
  • Low molecular weight compounds are about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, or about 1000 Daltons.
  • Chemotherapeutic agents contemplated for use include, without limitation, alkylating agents including: nitrogen mustards, such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); ethylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, c
  • epipodophylotoxins such as etoposide and teniposide
  • antibiotics such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycinC, and actinomycin
  • enzymes such as L-asparaginase
  • biological response modifiers such as interferon-alpha, IL-2, G-CSF and GM-CSF
  • miscellaneous agents including platinum coordination complexes such as cisplatin, Pt(IV) and carboplatin,
  • anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea,
  • methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine,
  • adrenocortical suppressants such as mitotane ( ⁇ , ⁇ ' -DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin- releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide.
  • mitotane ⁇ , ⁇ ' -DDD
  • hormones and antagonists including adrenocorticosteroid antagonists such
  • an architecture comprising a tocopherol modified oligonucleotide.
  • tocopherol is contemplated to be on the 5' end or the 3' end of an oligonucleotide or modified form thereof.
  • a tocopherol-modified oligonucleotide comprises a lipophilic end and a non-lipophilic end.
  • the lipophilic end comprises tocopherol, and may be chosen from the group consisting of a tocopherol derivative, alpha-tocopherol, beta-tocopherol, gamma-tocopherol and delta-tocopherol.
  • the lipophilic end in further embodiments, comprises palmitoyl, dipalmitoyl, stearyl, or distearyl.
  • the disclosure contemplates that cholesterol or phospholipids are used instead of tocopherol.
  • Cholesterol is attached in solid phase oligonucleotide synthesis, where it is mixed with the prepared liposomes to form SNAs.
  • liposomes composed of 95% 1 ,2-dioleoyl-sn-glycero-3 phosphatidylcholine (DOPC) and 5% 1 ,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6-azidohexanoyl) (DPPE-Azide) are prepared as described below. Then DBCO-modified oligonucleotides are added, which react with the azide lipid to functionalize the surface.
  • DOPC 95% 1 ,2-dioleoyl-sn-glycero-3 phosphatidylcholine
  • DPPE-Azide dipalmitoyl-sn-glycero-3-phosphoethanolamine
  • a phospholipid conjugated oligonucleotide is prepared as follows: First, a phosphatidylethanolamine lipid, such as DOPE, is reacted with succinimidyl 4- (p-maleimidophenyl)butyrate (SMPB) by mixing 25 mg/mL lipid, 1 equivalent SMPB and 1 equivalent of N,N-Diisopropylethylamine in chloroform. The mixture is reacted overnight. Next, the product is purified by flash chromatography using silica column (solvent A: dichloromethane, solvent B: methanol).
  • solvent A dichloromethane
  • solvent B methanol
  • the thiol-modified oligonucleotide (3' or 5' end modified) is reduced with 0.2M DTT and 0.1 M phosphate buffer (pH 8) at 40°C for 2 hours.
  • the oligonucleotide is then purified in a size exclusion column using water.
  • the phosphatidylethanolamine-SMPB lipid is dried over nitrogen gas and dissolved in ethanol in the same volume as the oligonucleotide.
  • the oligonucleotide is then mixed with the lipid such that the reaction is 50:50 water and ethanol. This mixture is reacted overnight, and the excess lipid is extracted by washing the reaction mixture with chloroform three times. Next, the aqueous phase and the interface are dried and dissolved in water. All lipid-conjugated oligonucleotides as disclosed herein are contemplated to be used interchangeably in the preparation of LSNAs.
  • the non-lipophilic end of the tocopherol-modified oligonucleotide is an oligonucleotide.
  • the oligonucleotide is either RNA or DNA.
  • the RNA can be an inhibitory RNA (RNAi) that performs a regulatory function, and is chosen from the group consisting of a small RNAi that is selected from the group consisting of a small inhibitory RNA (siRNA), an RNA that forms a triplex with double stranded DNA, and a ribozyme.
  • the RNA is microRNA that performs a regulatory function.
  • the RNA is a piwi-interacting RNA (piRNA).
  • the DNA is, in some embodiments, an antisense-DNA.
  • Oligonucleotides contemplated for use according to the disclosure are from about 5 to about 100 nucleotides in length. Methods and compositions are also contemplated wherein the oligonucleotide is 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 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 15 nucleotides in length, about 5 to about 10 nucleotides in length, and all oligonucleotides intermediate in length of the sizes specifically disclosed to the extent that the oligonucleot
  • oligonucleotides of 5, 6, 7, 8, 9, 10, 1 1 , 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, and 100 nucleotides in length are contemplated.
  • oligonucleotides include those containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain 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.”
  • Modified oligonucleotide backbones containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates,
  • phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters,
  • selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.
  • oligonucleotides having inverted polarity comprising a single 3' to 3' linkage at the 3'-most internucleotide linkage, i.e. a single inverted nucleoside residue which may be abasic (the nucleotide is missing or has a hydroxyl group in place thereof). Salts, mixed salts and free acid forms are also contemplated.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • oligonucleotide mimetics wherein both one or more sugar and/or one or more internucleotide linkage of the nucleotide units are replaced with "non- naturally occurring” groups.
  • this embodiment contemplates a peptide nucleic acid (PNA).
  • PNA compounds the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone. See, for example US Patent Nos. 5,539,082; 5,714,331 ; and 5,719,262, and Nielsen et al., 1991 , Science 254: 1497-1500, the disclosures of which are herein incorporated by reference.
  • oligonucleotides are provided with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and including— CH 2 — NH— O— CH 2 — ,— CH 2 — N(CH 3 )— O— CH 2 — classroom— CH 2 — O— N(CH 3 )— CH 2 — ,— CH 2 — N(CH 3 )— N(CH 3 )— CH 2 — and—0—N(CH 3 )—CH 2 —CH 2 — described in US Patent Nos. 5,489,677, and 5,602,240.
  • oligonucleotides with morpholino backbone structures described in US Patent No. 5,034,506.
  • Modified oligonucleotides may also contain one or more substituted sugar moieties.
  • oligonucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C 2 to Cio alkenyl and alkynyl.
  • FIG. 1 For embodiments, other embodiments include 0[(CH 2 ) n O] m CH 3 , 0(CH 2 ) n OCH 3 , 0(CH 2 ) n NH 2 , 0(CH 2 ) n CH 3 , 0(CH 2 ) n ONH 2 , and 0(CH 2 )nON[(CH 2 )nCH 3 ] 2 , where n and m are from 1 to about 10.
  • oligonucleotides comprise one of the following at the 2' position: Ci to Ci 0 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3 , OCF 3 , SOCH 3 , S0 2 CH 3 , ON0 2 , N0 2 , N 3 , NH2, heterocycloalkyl,
  • a modification includes 2'- methoxyethoxy (2 , -0-CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al., 1995, Helv. Chim.
  • the 2'-modification may be in the arabino (up) position or ribo (down) position.
  • a 2'-arabino modification is 2'-F.
  • oligonucleotide Similar modifications may also be made at other positions on the oligonucleotide, for example, at the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. See, for example, U.S. Pat. Nos. 4,981 ,957; 5,1 18,800; 5,319,080;
  • a modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.
  • the linkage is in certain aspects is a methylene (— CH 2 — ) n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2.
  • LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.
  • Oligonucleotides may also include base modifications or substitutions.
  • "unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified bases include other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, 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-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 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-trifluoro
  • Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine(1 H-pyrimido[5 ,4-b][1 ,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1 H-pyrimido[5 ,4-b][1 ,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine ⁇ e.g.
  • Modified bases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further bases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859,
  • 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1 .2°C. and are, in certain aspects combined with 2'-0-methoxyethyl sugar modifications. See, U.S. Pat. Nos. 3,687,808, U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273;
  • a "modified base” or other similar term refers to a composition which can pair with a natural base ⁇ e.g., adenine, guanine, cytosine, uracil, and/or thymine) and/or can pair with a non-naturally occurring base.
  • the modified base provides a T m differential of 15, 12, 10, 8, 6, 4, or 2°C. or less.
  • Exemplary modified bases are described in EP 1 072 679 and WO 97/12896.
  • nucleobase is meant the naturally occurring nucleobases adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) as well as non-naturally occurring nucleobases such as xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthine, 7-deazaguanine, N 4 ,N 4 -ethanocytosin, N',N'-ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C 3 — C 6 )- alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-tr- iazolopyridin, isocytosine, isoguanine, inosine and the "non-naturally occurring" nucleobases described
  • nucleobase thus includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Further naturally and non-naturally occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan, et al.), in Chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T. Crooke and B.
  • nucleosidic base or “base unit” is further intended to include compounds such as heterocyclic compounds that can serve like nucleobases including certain "universal bases” that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases.
  • universal bases are 3-nitropyrrole, optionally substituted indoles ⁇ e.g., 5-nitroindole), and optionally substituted hypoxanthine.
  • Other desirable universal bases include, pyrrole, diazole or triazole derivatives, including those universal bases known in the art.
  • oligonucleotide is exposed to the phosphoramidite-modified- tocopherol to create the tocopherol modified oligonucleotide.
  • any chemistry to one of skill in the art can be used to attach the tocopherol to the oligonucleotide, including amide linking or click chemistry.
  • the disclosure also provides methods of making liposomal particles.
  • a phospholipid, solvent, and a tocopherol modified oligonucleotide are provided. Then, the phospholipid is added to the solvent to form a first mixture comprising liposomes. The size of the liposomes in the first mixture is between about 100 nanometers and about 150 nanometers.
  • the liposomes are disrupted to create a second mixture comprising liposomes and small unilamellar vesicles (SUV).
  • the size of the liposomes and SUVs in the second mixture is between about 20 nanometers and about 150 nanometers.
  • the SUVs having a particle size between about 20 nanometers and about 50 nanometers are isolated from the second mixture. Finally, the tocopherol modified
  • oligonucleotide is added to the isolated SUVs to make a liposomal particle.
  • the particle size of the liposomal particles created by a method of the disclosure is less than or equal to about 50 nanometers.
  • a plurality of liposomal particles is produced and the particles in the plurality have a mean diameter of less than or equal to about 50 nanometers ⁇ e.g., about 5 nanometers to about 50 nanometers, or about 5 nanometers to about 40 nanometers, or about 5 nanometers to about 30 nanometers, or about 5 nanometers to about 20 nanometers, or about 10 nanometers to about 50 nanometers, or about 10 nanometers to about 40 nanometers, or about 10 nanometers to about 30
  • the particles in the plurality of liposomal particles created by a method of the disclosure have a mean diameter of less than or equal to about 20 nanometers, or less than or equal to about 25 nanometers, or less than or equal to about 30 nanometers, or less than or equal to about 35 nanometers, or less than or equal to about 40 nanometers, or less than or equal to about 45 nanometers.
  • the method comprises: (1 ) adding 1 X PBS to dry lipids to a final concentration of 1 -25 mg/mL (thus, in various embodiments, the final concentration is about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 mg/ml); (2) freezing rapidly in liquid nitrogen and thawing in a bath sonicator 3 times; (3) extruding through 200, 100, 80, 50 and 30 nm filters. Double filters are used and typically passed 2-10 times through each filter. In some embodiments, the process is stopped at 50 nm, but if 30 nm structures are desired, then the 30 nm filter is additionally added.
  • one probe sonicates after step (2).
  • the liposomes are centrifuged at 21000 x g for 10 minutes to remove metal shavings that come off in sonication and the mixture is extruded through a 30 nm filter as described in step (3).
  • the disclosure provides a method of making a liposomal particle, comprising adding a phospholipid to a solvent to form a first mixture, said first mixture comprising a plurality of liposomes; disrupting said plurality of liposomes to create a second mixture, said second mixture comprising a liposome and a small unilamellar vesicle (SUV); isolating said SUV from said second mixture, said SUV having a particle size between about 20 nanometers and 50 nanometers; and adding an oligonucleotide to the isolated SUV to make the liposomal particle.
  • SUV small unilamellar vesicle
  • an oligonucleotide associated with a LSNA inhibits the expression of a gene.
  • Methods for inhibiting gene product expression include those wherein expression of the target gene product is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% compared to gene product expression in the absence of a LSNA.
  • methods provided embrace those which results in essentially any degree of inhibition of expression of a target gene product.
  • the degree of inhibition is determined in vivo from a body fluid sample or from a biopsy sample or by imaging techniques well known in the art. Alternatively, the degree of inhibition is determined in a cell culture assay, generally as a predictable measure of a degree of inhibition that can be expected in vivo resulting from use of a specific type of liposomal SNA and a specific oligonucleotide.
  • a LSNA performs both a gene inhibitory function as well as an agent delivery function.
  • an agent is encapsulated in a LSNA as disclosed herein and the particle is additionally functionalized with one or more oligonucleotides designed to effect inhibition of target gene expression.
  • the methods include use of an oligonucleotide which is 100% complementary to the target polynucleotide, i.e., a perfect match, while in other aspects, the oligonucleotide is at least (meaning greater than or equal to) about 95% complementary to the polynucleotide over the length of the oligonucleotide, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20% complementary to the polynucleotide over the length of the oligonucleotide to the extent that the oligonucleotide is able to achieve the desired degree of inhibition of a target gene product.
  • an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
  • an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event ⁇ e.g., a loop structure or hairpin structure).
  • the percent complementarity is determined over the length of the oligonucleotide. For example, given an antisense compound in which 18 of 20 nucleotides of the antisense compound are complementary to a 20 nucleotide region in a target polynucleotide of 100 nucleotides total length, the oligonucleotide would be 90 percent complementary.
  • the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleotides.
  • Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and
  • the disclosure provides methods of utilizing LSNAs in gene regulation therapy.
  • This method comprises the step of hybridizing a polynucleotide encoding said gene product with one or more oligonucleotides complementary to all or a portion of said
  • polynucleotide said oligonucleotide being attached to a liposomal particle, wherein hybridizing between said polynucleotide and said oligonucleotide occurs over a length of said
  • the liposomal particle has a diameter that is about less than or equal to 50
  • the inhibition of gene expression may occur in vivo or in vitro.
  • the oligonucleotide utilized in this method is either RNA or DNA.
  • the RNA can be an inhibitory RNA (RNAi) that performs a regulatory function, and in various embodiments is selected from the group consisting of a small inhibitory RNA (siRNA), a single-stranded RNA (ssRNA), and a ribozyme.
  • RNAi inhibitory RNA
  • the RNA is microRNA that performs a regulatory function.
  • the DNA is, in some embodiments, an antisense-DNA.
  • Example 1 discloses the synthesis of a single liposomal spherical nucleic acid (LSNA) nanoparticle capable of dual inhibition of TLRs 4 and 9 ( Figure 1 ).
  • TLR4 inhibition will occur by encapsulation of TAK242, a small molecule inhibitor of TLR4.
  • TLR9 inhibition will occur by functionalization of the LSNA surface with an inhibitory CpG (iCpG) sequence called INH-18 (sequence: 5'-CCTGG ATGGG AACTTACCGCTGCA-3' ; SEQ ID NO: 1 ) ( Figure 2).
  • ICpG inhibitory CpG sequence
  • Lipid monomer 1 ,3-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and TAK-242 were dissolved in chloroform, combined in defined ratios and dehydrated to create a thin film. Lipids were rehydrated with a Tris buffer solution containing NaCI to create liposomes. Liposomes were probe-sonicated and membrane extruded to produce small unilamellar vesicles (SUVs). SUVs were passed through a sepharose size exclusion column, then particle size was measured by dynamic light scattering (DLS). To make the SUV an LSNA, tocopherol-anchored DNA was incubated with SUVs with agitation.
  • DOPC dioleoyl-sn-glycero-3-phosphocholine
  • TAK-242 Tris buffer solution containing NaCI
  • Liposomes were surface functionalized with oligonucleotides that bind the lipid membrane through a hydrophobic anchor (tocopherol or cholesterol).
  • a size shift from approximately 35nm to approximately 60nm indicates surface functionalization with oligonucleotides ( Figure 3).
  • the modularity and versatility of LSNA chemistry make this construct a desirable platform for delivery of therapeutic agents. Therefore, LSNAs have been designed for dual TLR inhibition by taking advantage of the properties of the liposome core and oligonucleotide shell.
  • the LSNAs were specifically designed to display TLR9-inhibitory DNA at the surface of the particle and encapsulate the small molecule TLR4-inhibitor, TAK-242 (ethyl(6R)-6-[N-(2-chloro-4- fluorophenyl) sulfamoyl]cyclohex-1 -ene-1 -carboxylate [(R)-(+)-5n), within the core membrane.
  • TAK-242 ethyl(6R)-6-[N-(2-chloro-4- fluorophenyl) sulfamoyl]cyclohex-1 -ene-1 -carboxylate [(R)-(+)-5n
  • TAK-242 was chosen as a TLR4 inhibitor because it binds the intracellular domain of TLR4 and interferes with binding of adaptor proteins, preventing signal transduction (li et al., Molecular pharmacology. 2006;69(4):1288-95; Kawamoto et al., European journal of pharmacology.
  • LSNAs are recognized by Scavenger Receptor A on the cell surface, which mediates endocytosis of the particle (Choi et al., Proceedings of the National Academy of Sciences of the United States of America. 2013;1 10(19)7625-30; Patel et al., Bioconjugate chemistry. 2010;21 (12):2250-6; Wu et al., Journal of the American Chemical Society. 2014;136(21 ):7726-33).
  • LC-MS liquid chromatography mass spectrometry
  • ICP-OES inductively- coupled plasma optical emission spectroscopy
  • Particles made from different molar ratios of lipid to TAK-242 have been shown to retain their small particle size and low polydispersity (Table 1 ). Once the particles are characterized, 3'- tocopherol is added to TLR9-inhibitory DNA, which anchors it into the liposomal bilayer.
  • RAW-Blue NF- ⁇ reporter mouse cell line
  • SEAP embryonic alkaline phosphatase
  • TLR9- or MPLA (TLR4)-stimulated RAW-Blue cells indicated that inhibition is specific to TLR4 activation by monophosphoryl lipidA (MPLA), and free INH-18 leads to inhibition of CpG- activated cells in a dose-dependent manner ( Figures 7 and 8). Notably, tests of free INH-18 activity showed a limited inhibition of MPLA activation ( Figure 7B) while exhibiting a high degree of inhibition of CpG-stimulated cells.
  • TLR activity was measured in HEK-Blue reporter cells using the secreted alkaline phosphatase (SEAP) assay. Briefly, cells engineered to express human or mouse TLRs are stimulated in response to their appropriate ligands. TLR activation leads to downstream activation of NF- ⁇ by alkaline phosphatase activation. AP1 is secreted into the media and converts it from pink to purple/blue ( Figure 4).
  • SEAP secreted alkaline phosphatase
  • Table 1 shows the composition and characterization of TAK-encapsulated liposomes.
  • FIG. 6 shows the uptake of dye-labeled LSNAs in mouse macrophages.
  • Figure 7 shows TLR inhibition by free INH-18 or TAK-242 is specific.
  • RAW-Blue macrophages were stimulated with either 1826 CpG DNA (TLR9 ligand) or monophosphory lipid A (MPLA, TLR4 ligand).
  • TLR9 inhibition is clearly specific to INH-18 oligonucleotide (A), but TLR4 inhibition, though mostly inhibited by TAK-242, is slightly inhibited by INH-18 (B). Crosstalk between TLR4 and TLR9 pathways could explain this phenomenon.
  • Table 2 shows the uptake of dye-labeled LSNAs in mouse macrophages.
  • FIG. 2 shows TLR inhibition by free INH-18 or TAK-242 is specific.
  • RAW-Blue macrophages were stimulated with either 1826 CpG DNA (TLR9 ligand) or monophosphory lipid A (MPLA, TLR4 ligand).
  • FIG. 8 shows potent TLR4 and TLR9 inhibition by LSNAs in mouse and primary human cells.
  • Four different LSNAs were synthesized and RAW-Blue cells were treated with 500nM LSNAs to test for ability to modulate TLR activation (A-B).
  • A-B Only particles containing INH-18 inhibitory sequences are capable of suppressing TLR9 activity (A) and more potently than free INH-18 DNA, while particles containing TAK-242 suppress TLR4 activity (B) comparable to free TAK-242.
  • CpG-induced human peripheral mononuclear blood cell (hPBMC) proliferation is inhibited in a dose dependent manner when treated with dual-targeting LSNAs (C).
  • This example demonstrates successful synthesis and in vitro characterization of a liposomal nanoparticle that can encapsulate a hydrophobic small molecule inhibitor of TLR4 and features TLR9 inhibiting oligonucleotides on the surface.
  • This construct was rapidly taken up by murine macrophages and effectively suppressed downstream activation of NF- ⁇ . It also diminished human lymphocyte proliferation.
  • the construct was tunable for TLR9 suppression by altering the backbone of the oligonucleotides from phosphodiester to phosphorothioate.
  • TLR9 inhibiting sequences will be screened and the backbone chemistry will be varied.
  • the dose and time dependent response of RAW-Blue and HEK-Blue reporter cells induced with MPLA and CpG DNA simultaneously to dual TLR-targeting LSNAs will also be examined. Further, downstream inhibition of cytokines will be measured using human PBMCs. Ultimately, this dual-targeting particle will be tested in an ischemia reperfusion/transplantation mouse model.
  • TLRs Toll-like receptors
  • TLRs autoimmune disorders, sepsis and ischemia reperfusion injury (IRI).
  • IRI ischemia reperfusion injury
  • TLR4 inhibition occurs by encapsulation of a small molecule (TAK-242).
  • TLR9 inhibition occurs by functionalization of the LSNA surface with an inhibitory DNA sequence (INH-18) ( Figure 10).
  • Lipid monomer 1 ,3-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and TAK-242 were dissolved in chloroform, combined in defined ratios and dehydrated to create a thin film. Lipids were rehydrated with a Tris buffer solution containing NaCI to create liposomes. Liposomes were probe sonicated and membrane extruded to produce small unilamellar vesicles (SUVs). SUVs were passed through a sepharose size exclusion column, then particle size was measured by dynamic light scattering (DLS). To make the SUV into an LSNA, cholesterol- anchored DNA was incubated with SUVs at 37°C with agitation.
  • DOPC dioleoyl-sn-glycero-3-phosphocholine
  • TAK-242 Tris buffer solution containing NaCI
  • TAK-242 encapsulated was measured by sulfur analysis using inductively coupled plasma optical emission spectrometry (ICP-OES) and confirmed by liquid chromatography Mass spectrometry (LC-MS).
  • TLR activity was measured in HEK-Blue reporter cells using the secreted alkaline phosphatase (SEAP) assay. Briefly, human cells engineered to express mouse TLRs were stimulated with their appropriate ligands. TLR activation leads to downstream activation of NF- ⁇ and subsequent alkaline phosphatase translation. AP1 is secreted into the media and converts the media from pink to purple/blue. See Figures 3 and 4.
  • FIG. 1 1 shows potent TLR4 and TLR9 inhibition by LSNAs in engineered TLR reporter cells.
  • Four different LSNAs were synthesized and HEK-Blue cells expressing mouse TLR4 or mouse TLR9 were treated with LSNAs (125nM by DNA, 75nM by TAK) to test for the ability to modulate TLR activation (A-B).
  • Only particles containing INH-18 inhibitory sequences were capable of suppressing TLR9 activity (A), while particles containing TAK-242 suppressed TLR4 activity (B).
  • Figure 12 demonstrates the kinetic advantage of using LSNAs.
  • the speed at which TLR9 activity was suppressed was assessed using HEK-Blue cells expressing mouse TLR9. Cells were treated for 4 hours simultaneously with agonist and antagonists (A) or with a 4 hour pulse of agonist followed by 8 hours with antagonist (B).
  • Figure 13 demonstrates that LSNAs are potent inhibitors of pro-inflammatory cytokine production in primary mouse cells.
  • Primary mouse splenocytes were isolated and treated with 500nM TLR9 agonist (Figure 13A-E) or 500ng/ml TLR4 agonist ( Figure 13F) to stimulate cytokine production. In general, dose dependence of antagonist was observed.
  • IRI Ischemia Reperfusion Injury
  • Figure 15 shows results of a liver function test and IL-6 levels following IRI.
  • ALT alanine transaminase
  • ALP alkaline phosphatase
  • TBIL total bilirubin
  • CTL cholesterol
  • proinflammatory cytokine IL-6 proinflammatory cytokine IL-6.
  • the nanoparticle core which is used to encapsulate the TLR4 antagonizing small molecule drug TAK-242 (i.e., Resatorvid; CLI-095), is comprised to lipid monomers.
  • DOPC is the lipid monomer most commonly used due to it being an FDA approved material, but other lipids may be incorporated to impart chemical features and/or biological effects.
  • the outer surface of the lipid core is decorated with synthesized DNA that has a hydrophobic moiety conjugated to it.
  • the hydrophobic moiety inserts into the lipid bilayer of the nanoparticle core and anchors the DNA to the outer surface.
  • the DNA sequence chosen is ODN INH-18, which is a TLR9 inhibitor specific to mouse and human TLR9. Other TLR9 targeting sequences were screened, but INH-18 was chosen for its potency.
  • TLR4 inhibiting small molecule drug (TAK-242 (i.e., Resatorvid) is made by dissolving the TAK-242 in chloroform. This is mixed with lipid monomers that come dissolved in chloroform, and the solvent is evaporated under nitrogen. The lipid films are dried overnight using lyophilization. The dried lipid films are rehydrated with buffer and probe sonicated, ultracentrifuged, and extruded to obtain uniformly sized liposomes containing TAK. Any unencapsulated material is removed using size exclusion. See Figure 16.
  • the size and polydispersity index (PDI) are determined using dynamic light scattering (DLS).
  • DLS dynamic light scattering
  • ICP-OES inductively coupled plasma-optical emission spectrometry
  • Figure 18 shows TLR9 suppression in HEK-Blue cells expressing mouse TLR9.
  • FIG 19 shows TLR4 suppression in HEK-Blue cells expressing mouse TLR4.
  • Figure 20 shows TLR9 suppression in HEK-Blue cells expressing mouse TLR9.
  • This example demonstrates that immuno-stimulatory SNAs (IS-SNAs) are taken up by dendritic cells in lymph nodes. More specifically, this example demonstrates the use of encapsulated spherical nucleic acid (SNA) vaccines. In this example, the encapsulated payload was tumor-specific antigen. See Figure 21 .
  • Engineered reporter cell lines as well as primary cells are used as tools to measure NF-KB activation, cell proliferation, and cytokine production as a readout of TLR activity. Results from these studies will provide insight and link oligonucleotide chemistry to the cellular response elicited by LSNAs and the synergistic effect of combining two TLR-inhibitory strategies in a single nanoparticle.
  • TLR9 expression varies between mouse and human cells, and this may result in differences in potency of TLR9 inhibition by LSNAs (Fitzner et al., International immunopharmacology.
  • PBMCs stimulated with MPLA and/or CpG.
  • PBMCs play a role in the propagation of the inflammatory response and the cell-based immunologic response to IRI. These cells are recruited to the reperfused, transplanted organ, proliferate, and facilitate death of cells within the tissue and release inflammatory signals.
  • tritium (3H)-labeled thymidine incorporation is used as a measure of LSNA inhibitory activity against activated PBMCs.
  • Cells are stimulated with the appropriate ligands for TLR4 or TLR9 (MPLA or stimulatory CpG DNA, respectively) or with polyhemagglutinin, a plant mitogen, as a control.
  • TLR4 or TLR9 MPLA or stimulatory CpG DNA, respectively
  • polyhemagglutinin a plant mitogen
  • TLR signaling pathways involve multiple molecular mediators that ultimately result in the production of cytokines and chemokines.
  • Classical mediators of a pro-inflammatory response in stimulated PBMCs include IL-1 A, IL-1 B, IL-2, IL-4, IL-6, IL-12, TNFa, IFNy, and GM- CSF.
  • IL-1 A IL-1 A
  • IL-1 B IL-1 B
  • IL-2 IL-2
  • IL-4 IL-6
  • IL-12 TNFa
  • IFNy IFNy
  • GM- CSF GM- CSF
  • Quantitative real-time reverse transcription polymerase chain reaction and Western blot are used to analyze TLR4 and TLR9 mRNA and protein expression and mRNA and protein expression and phosphorylation levels of TLR signal transduction mediators; such as MyD88, TRIF and TRAF3 (MyD88 independent pathway), JNK and p38 MAPK, and NF-KB.
  • TLR signal transduction mediators such as MyD88, TRIF and TRAF3 (MyD88 independent pathway), JNK and p38 MAPK, and NF-KB.
  • DOPE 1 ,2-dioleoyl-sn-glyvero-3-phophoethanolamine
  • DOPE may increase release of TAK-242 and entry into the cytoplasm.
  • DOPE like DOPC, is a neutral lipid (as opposed to cationic or anionic), but DOPE is pH-sensitive. At lower pH, as found in the endosome, DOPE undergoes a conformational change that may facilitate core fusion with the endosome and increased endosomal escape of TAK-242 (Bergstrand et al., Biophysical chemistry.
  • the optimal time course and approach for LSNA administration in a liver transplantation model is determined by comparing the effects of pretreatment of the donor animal prior to liver harvest to pretreatment of the liver while it is in cold preservation solution containing LSNAs, and to intravenous treatment of the recipient animal at the time of transplantation.
  • the results of this study will inform on the timing of treatment when transplantation IRI damage is most responsive to LSNA treatment.
  • the expectation is that treatment with dual-targeting LSNAs will reduce IRI-induced damage to a transplantable organ with the greatest efficacy of treatment obtained by administering LSNAs prior to the harvest of the donor liver.
  • LSNAs are administered to donor mice 30 minutes prior liver removal, in cold University of Wisconsin preservation solution for an hour at 4° C, or intravenously to recipient syngeneic mice at the time of transplantation. Blood samples are collected at 30 minutes, 24 hours, and 72 hours following transplantation or sham surgery (control). These samples are tested for markers of liver damage: ALT (alanine aminotransferase), AST (aspartate aminotransferase) and AlkPhos (alkaline phosphatase). Bilirubin levels will also be compared between treated transplant recipient animals and control animals given non-targeting, inert LSNAs (Scheig, Primary care. 1996;23(3):551 -60).
  • the Luminex system is used to quantify the production of classical pro-inflammatory markers, specifically IL-1 A, IL-1 B, IL-2, IL-4, IL- 6, IL- 12, TNFa, IFNy, and GM-CSF, in collected blood samples.
  • classical pro-inflammatory markers specifically IL-1 A, IL-1 B, IL-2, IL-4, IL- 6, IL- 12, TNFa, IFNy, and GM-CSF
  • IRI can be induced by hilar clamping of the portal vein and hepatic artery that supplies blood to the liver, thereby eliminating the complexity of the liver transplantation model and reducing the number of animals needed.
  • hilar clamping does not recapitulate the clinical scenario of liver transplantation and the effects of the cold ischemia component on IRI.
  • it removes the ability to evaluate ex vivo treatment of the liver graft, which is an attractive route for direct treatment of the graft and avoids the pharmacological challenges associated with systemic LSNA administration.
  • liver graft tissue recovered from necropsy at 72 hours will be analyzed for proteoform frequency, which will be correlated with liver function (ALT, AST), recipient survival and LSNA treatment regimen.
  • the goal is to develop an initial biomarker signature to correlate severity of liver IRI and response to LSNA treatment.
  • TLR4 or TLR9 knockout mice are used as either donors or recipients along with wild-type animals in studies using mono-inhibitory LSNAs. As only one population of animals (donor or recipient) will express the wild-type TLRs, any inhibitory effects seen from LSNA treatment are attributed to cells originating from the wild-type animal.
  • SNAs are synthesized by adding single-stranded RNA to liposomes for 4 hours at 37°C while shaking.
  • Liposomes are constructed from 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a biocompatible FDA approved material, using sonication and extrusion extruded through a 30 nm membrane. Concentration of liposomes are quantified using an ICP-OES for phosphorous.
  • DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
  • Concentration of liposomes are quantified using an ICP-OES for phosphorous.
  • single-stranded RNA containing a hydrophobic anchor tocopherol or cholesterol
  • was synthesized using an RNA synthesizer processed using recommended procedures on the Glen Research website and purified using reverse phase HPLC (see below for more details pertaining to sequence).
  • the ratio of lipid monomer to RNA upon mixing is 100 to yield approximately
  • FBS fetal bovine serum
  • LSNAs' mode of entry is through endocytosis by caveolin-mediated lipid rafts, and traffic through the endocytic pathway. Confocal results indicate that LSNAs co- localize with late endosomal and lysosomal markers within 45 minutes of transfection.
  • TLR7/8 expressing reporter cell lines human embryonic kidney 293 engineered cells (Hek-Blue) that are engineered to contain either TLR7 or TLR8 and an inducible secreted alkaline phosphatase (SEAP) upon NF- ⁇ activation
  • Relative activity of TLR7/8 activation was quantified using a QuantiBlue Assay (through the formation of NBD diformazan and an indigo dimer).
  • Figure 28 shows that LSNAs exhibit enhanced activation of TLR7/8 over free RNA, and RNA transfected with a cationic transfection agent.
  • RNA Backbone phosphothiolate and phosphodiester backbones, where phosphothiolate backbone is expected to increase the stability and perhaps the uptake of LSNAs into cells
  • Sequence length (increasing and decreasing the length of the RNA sequence by increasing the number or repeats in the sequence
  • the most potent sequence tested for activating TLR7 was "cholesterol - UGU CCU UCA AUG UCC UUC AA” (SEQ ID NO: 10) while the most potent sequence tested for activating TLR8 was "cholesterol - PEG spacer - UGU CCU UCA AUG UCC UUC AA” (SEQ ID NO: 1 1 ), all with a phosphothiolate backbone. Further studies will explore the role of oligonucleotide density in determining TLR7/8 activity in Hek-Blue reporter cells.
  • LSNAs induce dendritic cell maturation by upregulating co-stimulatory molecules CD80 and CD86 (CD86+, CD1 1 c+ shown below) on C57BL/7 dendritic cell after a 24 hour incubation at 37°C at a cell density of 400,000 cells/mL. After incubation, cells were stained with fluorescent antibodies against CD80, CD86, and CD1 1 c, and fixed in 0.5%
  • Resiquimod a TLR7/8 agonist that was used as a positive control ( Figure 29).
  • the upregulation of co-stimulatory markers is crucial for dendritic cells to activate T-cells.
  • LSNAs activate antigen presenting cells
  • they were introduced to dendritic cells (1 ,000,000 cells/mL for 48 hours at 37°C), B cells (1 ,000,000 cells/mL for 24 hours 37°C), and splenocytes (1 ,000,000 cells/mL 37°C) from C57BL/6 mice.
  • Dendritic cells were directly purchased, while B cells were isolated using an enrichment kit from C57BL/7 splenocytes. After the incubation period, cell supernatants were isolated and analyzed using magnetic bead immunoassays and quantified using the Luminex.
  • LSNAs induce the production of IL-6 (released in late endosomes) more so than free RNA, a control SNA, or untreated ( Figure 30). This coincides with the localization of LSNAs (in the endolytic pathway).
  • levels of IL-2 and IFN-y were elevated in splenocytes treated with LSNAs compared to untreated, free RNA or control LSNAs.
  • the next step in this study is to co-deliver an antigen alongside the RNA agonists on the same nanoparticle and study antigen presentation in dendritic cells.
  • the rate of antigen presentation (peptides expressed on MHC class II receptors) is analyzed using flow cytometry while cytokine activity after antigen presenting cells transfected with LSNA cancer vaccines is quantified.
  • LSNAs function as effective therapeutic cancer vaccines, they must (1 ) bind and activate TLR7/8 selectively, (2) activate dendritic cells, (3) present tumor antigen on cell surfaces, and (4) induce cytotoxic T-cell mediated apoptosis of tumor cells.
  • TLR7/8 HEK- Blue reporter cell lines that stably express TLR7 and TLR8 (A). They have also been shown to maturate dendritic cell through the upregulation of CD80/CD86 co-stimulatory markers as analyzed by flow cytometry (B).
  • LSNAs induce cytokine secretion (IL-6 upregulation in C57BL/6 B cells, dendritic cells splenocytes as well as IL-2 and I FN- ⁇ secretion C75BL/6 splenocytes (C).
  • IL-6 upregulation in C57BL/6 B cells dendritic cells splenocytes as well as IL-2 and I FN- ⁇ secretion C75BL/6 splenocytes (C).
  • C I FN- ⁇ secretion C75BL/6 splenocytes
  • LSNA TLR7/8 agonists can be used to activate and mature dendritic cells in a potent and selective manner. Experiments elucidating the mechanism of immune cell activation by studying downstream activation and maturation of T-cells, B cells, and NK cells are being explored. In addition, tumor-associated antigens are being encapsulated in the liposomal core of the LSNA to probe antigen presentation on dendritic cells and cytotoxic T- cell mediated cell death in tumor cells. Lastly, these LSNA cancer vaccine formulation are being tested in vivo to confirm their tumor cell lysis efficacy.
  • OVA-I Peptides SIINFEKLC (SEQ ID NO: 13), CSIINFEKL (SEQ ID NO: 14), Alexa Fluor 488 and FITC labeled OVA-I
  • the method used for preparing the liposomes was as described herein above.
  • the peptide or protein was added into the 1 X PBS that was used for rehydrating the lipids. After freeze/thaw cycles and extrusion, excess lipid was removed by dialyzing against 1 X PBS with a suitable dialysis membrane (typically 10K for peptides, 100K for proteins). Quantification of DOPC in the liposomes
  • RNA was synthesized on a MerMade 12 using phosphoramidites from ChemGenes: ssRNA DR (5'-gcc cga cag aag aga gac ac-3' (SEQ ID NO: 5) and ssRNA-41 (5'-ugu ecu uca aug ucc uuc aa-3' (SEQ ID NO: 6)), all with a phosphothiolate backbone. More detailed sequence information for RNA used in this paper are located in the Table 5. Cy-3.5 modified sequences were used for all uptake experiments (Figure 36); functional experiments were conducted with non-dye-labeled oligonucleotides. Standard oligonucleotide downstream processing of oligonucleotide sequences was conducted according to Glen Research's recommended protocols.
  • Cy-3.5 labeled cholesterol-modified RNA contain an internal Cy-3.5 adjacent to the cholesterol, while Cy3.5 labeled RNAs without cholesterol contain a dTdT (phosphate backbone) 5' modification to prevent the assembly of micelles.
  • dTdT phosphate backbone
  • ,2-dioleoyl-sn-glycero-3-phosphocholine were purchased from Avanti Polar Lipids, dried into a thin film, and hydrated using a solution containing a model peptide antigen, SIINFEKL (SEQ ID NO: 12) (1 mg/mL) dissolved in 30mM HEPES buffer and 0.15M NaCI.
  • SIINFEKL SEQ ID NO: 12
  • liposomes were underwent freeze/thawed in LiqN 2 /RT and manually extruded through a 200, 100, 80, 50 and 30 nm filter for 10 passes each.
  • the liposomes were then purified using tangential flow filtration module (30k pore size) with 20x excess HEPES/Saline buffer to remove the excess peptides that were not encapsulated in the liposomes.
  • concentration of liposomes in solution was determined by relating the phosphate content as determined using ICP-OES [Banga et al., J Am Chem Soc 2014, 136 (28), 9866-9].
  • liposomes were functionalized with RNA at a ratio of 1 :80 (RNA to lipid monomer) and shaken overnight at room temperature.
  • LSNAs Size and charge of LSNAs were measured using DLS (Malvern). To determine the functionalization efficiency of LSNAs, 10pmol of Cy3.5 labeled RNA and LSNA was loaded onto an agarose gel (1 %) and run at 80V for 45 minutes on ice and imaged. The percentage of free oligonucleotides was determined using band intensity analysis on ImageJ, resulting in approximately 12% free cholesterol and Cy3.5 labeled RNA. Nanoparticle loading
  • Reporter cells engineered to express TLR7/8 were purchased from Invivogen and cultured according to the manufacturer's protocols.
  • PBMCs Primary mouse splenocytes (both C57/BL6 WT and OT-1 ) were gifted from the Bin Zhang Laboratory. Human PBMCs were purchased from Zen-Bio. All primary cells were cultured in RPMI containing 10% FBS and 1 % Pen-Strep.
  • QuantiBlue Invivogen for 1 hour to detect secreted embryonic alkaline phosphatase (SEAP), a measure of NF- ⁇ activation.
  • SEAP embryonic alkaline phosphatase
  • Bone marrow derived dendritic cells were incubated with Cy-3.5 labeled ssRNA in free, cationically transfected, or LSNA form at a concentration of 1 urn at 24 hours. Cells were washed with PBS and fixed with 3.7% paraformaldehyde. Cells were then resuspended in VectaShield Mounting Medium with DAPI and fixed on a slide. All images were taken on a Zeiss LSM800 using a 63x oil objective.
  • Murine splenocytes (seeded at 1 E6 cells/mL) were incubated with Cy-3.5 labeled ssRNAs in free, cationically transfected, or LSNA form for both 8 hours and 24 hours.
  • Murine splenocytes were seeded at a density of 5x10 6 cells/well and treated with constructs containing 1 uM of RNA (and 1 uM for resiquimod). Cytokine release was quantified using magnetic bead assays kits from either eBioscience, R&D Biosystems, or Millipore and analyzed on a Luminex 200, all according to each manufacturer's instructions. Various dilutions of cell culture supernatants (from no dilution to 10x dilution) were used to ensure the
  • murine splenocytes were seeded at a concentration of 1 x10 6 cells/mL and treated with constructs (1 uM by RNA) for 24 hours. Cells were then washed and stained with the following antibodies for lineage: pDCs (B220 + , PDCA + , CD1 1 c med ), B cell (B220 + ), macrophages (F4/80 + , CD1 1 b + ), NK (NK1 .1 + , CD49b + , CD3 " ) and T cells (CD3 + ).
  • Cells were also stained with CD86, MHCII, and CD69 to measure activation and H-2kb/SIINFEKL(SEQ ID NO: 12) for antigen presentation. All samples were analyzed using an LSRII. All antibodies were purchased from either BD Biosciences, eBioscience, or Biolegend.
  • DCs primed with all constructs (1 uM by RNA, 1 uM resiquimod) were seeded at 0.5E6 cells/mL for 24 hours before they were washed and co-cultured with positively selected CD8+ OT-1 T cells (EasySepTM Mouse CD8+ T Cell Isolation Kit from STEMCELL Technologies) at a 1 :1 ratio (50k cells DCs : 50k OT-1 T cells to reach a total density of 1 E6 cells/mL) for 72 hours.
  • CD8+ OT-1 T cells EasySepTM Mouse CD8+ T Cell Isolation Kit from STEMCELL Technologies
  • 50k cells DCs : 50k OT-1 T cells to reach a total density of 1 E6 cells/mL for 72 hours.
  • cells were washed and stained using the antibodies against CD8 and CD90.1 .
  • the membrane was then permeated with a FOXP3 permeation kit and stained with KI-67. All samples were analyzed using
  • CCR2 CCR2, CC chemokine receptor 2; CCL2, CC chemokine ligand 2; CCR5, CC chemokine receptor 5; TLC, thin layer chromatography.
  • IS-r-LSNAs functionalized with ssRNA range from 70-75 nm in diameter ( Figure 34B), with a size increase of approximately 10nm over bare liposomes, as determined using DLS.
  • the functionalization of RNA to bare liposomes can be also be visualized via agarose gel, where bands denoting Cyanine 3.5 (Cy3.5) labeled RNA functionalized LSNAs have lower mobility compared 5'cholesterol and Cy3.5 modified RNA (Figure 34C).
  • Cy3.5 Cyanine 3.5
  • An increase in RNA stability can also be seen on IS-r-LSNAs compared to that of free RNA, where degradation products of free RNA appear after a 1 -hour incubation in 10% FBS, while RNA isolated from LSNAs remain intact. CryoEM images were also captured (Figure 40).
  • r-LSNA DR ssRNA LSNA TLR7/8 agonist
  • r-LSNA 41 control ssRNA LSNA
  • free DR ssRNA TLR7/8 agonist only
  • LyoVec DR ssRNA TLR7/8 agonist admixed with cationic transfection agent
  • IS-r-LSNAs are 3x more potent in activating TLR7 than free RNA or cationic transfected RNA, while it is 12x and 3x more potent in activating TLR8 than free RNA or cationic transfected RNA, respectively.
  • Figure 41 shows data obtained during the probing of the structure/function relationship between TLRs 7/8 and IS-r-LSNAs.
  • Figure 41 A shows that several sequences were screened to identify the optimal sequence that was most potent in activating TLR7/8.
  • the potency of five different sequences was screened by quantifying NF-KB activity in Raw Blue cells (Invivogen).
  • the most potent sequence was chosen for all further downstream experiments.
  • To determine the optimal backbone composition for ssRNA-DR free, cationically transfected or LSNA DR were synthesized with either a
  • Cy-3.5-labeled free RNA can be attributed to either to the phosphothiolate backbone, which allows it to enter cells [Overhoff et al., EMBO Rep 2005, 6 (12), 1 176-81 ; Koller et al., Nucleic Acids Res 201 1 , 39 (1 1 ), 4795-807; Moseman et al., J Immunol 2013, 191 (1 1 ), 5615-24] or via non-specific phagocytosis by the BMDCs.
  • mice splenocytes were treated with ssRNA in free, cationically transfected, and LSNA form ( Figure 36B).
  • Various immune cell populations were separated using flow cytometry analysis through their cell surface phenotypic markers: pDC (B220 + , CD1 1 c med , CD19 " ), cDC (CD1 1 c + , CD19 " , B220 “ ) (CD19 + , B220 + ), NK ( ⁇ 1 . ⁇ , CD49b + , CD3 " ), macrophage (CD1 1 b + , F4/80 + ), and granulocyte (Gr1 + ).
  • LSNAs exhibit higher uptake into cells than free RNA or cationically transfected RNA in pDCs, macrophages, and NK cells.
  • uptake of LSNAs in pDCs at 8h is the highest compared to any other cell type, and is 2-5x higher than that of free ssRNA or cationically transfected ssRNA.
  • Figure 42 shows results of studies examining the viability of all constructs in Hek 293 cells expressing either TLR7 or 8 as determined using an Alamar Blue assay after a 24 hour incubation. While free RNA or rLSNAs do not decrease viability, the use of a cationic transfection agent LyoVec does decrease cell viability. Figure 43 shows that overnight incubation of Hek-Blue cells with chloroquine does not decrease cell viability. Stimulatory Activity of IS-r-LSNAs in Primary Immune Cells
  • IS-r-LSNAs induced significantly higher levels type I IFNs (mostly secreted from plasmacytoid dendritic cells, as they can secrete 100x more type I IFNs as compared to other APCs upon TLR7 activation [Reizis et al., Annual review of immunology 201 1 , 29, 163-83; Hornung et al., Nat Med 2005, 1 1 (3), 263-70; Gary-Gouy et al., J Interferon Cytokine Res 2002, 22 (6), 653-9; Gilliet et al., Nat Rev Immunol 2008, 8 (8), 594- 606]) than that of free RNA. While the secreted levels of IFN.
  • IS-r-LSNAs also induce other pro-inflammatory cytokines, such as IL-6, IL10, IP-10, IL-12p40, and TNFa, which are secreted from many immune cell types: DCs, B cells, macrophages, T cells, monocytes, mast cells, and granulocytes [Lacy et al., Blood 201 1 , 1 18 (1 ), 9-18; Stanley et al., Physiology (Bethesda) 2010, 25 (4), 218-29; Couper et al., J Immunol 2008, 180 (9), 5771 -7; Liu et al., Cytokine Growth Factor Rev 201 1 , 22 (3), 121 -30; Hamza et al., Int J Mol Sci 2010, 1 1 (3), 789-806],all of which are higher than free RNA and control LSNAs.
  • FIG. 46 shows the results of a comparison between ssRNA-DR as functionalized onto r-LSNAs or cationically transfected for cytokine secretion.
  • IS-r-LSNAs To determine whether IS-r-LSNAs can specifically maturate DCs, all constructs were incubated with positively selected (CD1 1 c + ) DCs for 24 hours, and analyzed using flow cytometry to look at the DC maturation through the upregulation of co-stimulatory and MHC-II receptors (Figure 39A).
  • IS-r-LSNAs upregulate CD86 and MHCII receptors on pDCs (B220 + , CD1 1 c med ) compared to control LSNAs, free RNA, and cationically transfected ssRNA, an important step in the DC maturation process (Figure 45).
  • IS-r-LSNAs To pinpoint which cell types IS-r-LSNAs can activate, all constructs were incubated with a mixed pool of primary immune cells (murine splenocytes) for 24 hours. Flow cytometry was used to identify various immune cell types and to evaluate each cell's ability to induce a second signal of APC activation: upregulation of co-stimulatory and MHC-II receptors (Figure 38B). As expected, IS-r-LSNAs show increased CD86 and MHCII receptor expression on pDCs (PDCA + , CD1 1 c med , B220 + ) compared to free ssRNA and on-par if not better than cationically transfected ssRNA.
  • IS-r-LSNAs also induce maturation two other types of APCs: B cells (B220 + ) and macrophages (F4/80 + , CD1 1 b + ) via CD86 and MHCII receptor upregulation compared to free ssRNA or cationically transfected ssRNA.
  • B cells B220 +
  • macrophages F4/80 +
  • CD1 1 b + macrophages
  • MHCII receptor upregulation compared to free ssRNA or cationically transfected ssRNA.
  • IS-r-LSNAs also activate NK (NK1 . ⁇ , CD49b + , CD3 " ) and T cells (CD3 + ), as indicated by upregulation of CD69 when compared to free ssRNA.
  • IS-r-LSNAs within the liposome core, IS-r-LSNAs are able to activate many types of immune cells, including APCs while precisely co-delivering antigen to the same cell. This is key, as the delivery of antigen without the without presence of an immunogenic adjuvant can induce tolerance [O'Neill et al., Nat Rev Immunol 2013, 13 (6), 453-60; Mahnke et al., Immunol Cell Biol 2002, 80 (5), 477-83; Dudek et al., Front Immunol 2013, 4, 438].
  • SIINFEKL(SEQ ID NO: 12) peptide for 24 hours.
  • the presentation of SIINFEKL(SEQ ID NO: 12) on MHC-I receptors for each immune cell was determined using an antibody that binds the SIINFEKL(SEQ ID NO: 12)-MHC-I complex (H-2Kb-SIINFEKL (SEQ ID NO: 12), eBioscience), quantified using flow cytometry ( Figure 39A).
  • IS-r-LSNAs increase antigen presentation rates in APCs, particularly in DCs, which see the highest expression of MHC-I bound SIINFEKL(SEQ ID NO: 12) after treatment by IS-r-LSNAs compared to free or cationically transfected ssRNA.
  • IS-r-LSNAs immunomodulatory spherical nucleic acids
  • the IS-r-SNAs consist of a 65nm liposomal core decorated with TLR7/8 agonist RNA strands. These constructs demonstrate high cellular uptake and potent modulation of endosomal TLR7/8 in multiple reporter cell lines and primary cells in vitro.
  • IS-r- LSNAs induce activation of APCs and also NK and T cells, through increased cytokine secretion and co-stimulatory receptor upregulation.
  • IS-r-LSNAs also allowed the co-delivery of a peptide antigen within the same construct, thus increasing antigen presentation rates in APCs, resulting in an enhanced antigen-specific CD8+ T cell response in OT-1 cells when compared to free or cationically transfected ssRNA.
  • TNBC Triple Negative Breast Cancer
  • SNAs were tested in the 4T1 syngeneic tumor mouse model of TNBC for their ability to activate the immune system and initiate an antitumor response against established TNBC mammary tumors. Mice were vaccinated twice, five days apart, after the tumors had already started growing to mimic the clinical situation where a patient would being treatment after the tumors are detected. This treatment regime enabled a 30% reduction in the final tumor volume compared with untreated tumor-bearing animals. There was also double the number of T-cells in the tumor microenvironment suggesting the SNA vaccine was successful in turning on the immune system and recruiting active T-cells to the tumor site. Additionally, the SNA vaccine treatment greatly reduced the number of lung metastases (Figure 50), which is highly significant as metastatic tumors are notoriously more difficult to treat than the primary breast tumor
  • Additional strategies contemplated by the disclosure include increasing the vaccination dose, targeting different TLRs, or combining SNAs with other immunotherapies such as checkpoint inhibitors [Postow et al., J. Clin. Oncol. 33, 1974-1982 (2015)].
  • Liposomes were formed by five freeze-thaw cycles freeze-thaw cycles in liquid nitrogen and a 37oC water bath followed by ten rounds of high-pressure extrusion through polycarbonate filters with pore sizes of 200, 100, 80, and 50 nm. Tangential flow filtration with a pore size of 500 kDa was used to remove any non-encapsulated proteins and the sample was repeatedly washed with phosphate buffered saline until no protein was detected in the flow-through, as monitored by measuring the absorption of the flow-through at 280 nm with ultraviolet-visible spectroscopy.
  • the amount of protein encapsulated within the liposomes was measured using the BCA assay after disruption of the liposome with 1 % SDS to release the encapsulated protein.
  • the phospholipid concentration was measured using a phosphatidylcholine quantification kit.
  • Cholesterol terminated oligonucleotides were attached to the surface of the liposomes by mixing 20 ⁇ oligonucleotides with a solution of liposomes at 1 .63 mM lipid at 25°C overnight.
  • the oligonucleotide concentration was determined by measuring the absorption at 260 nm with ultraviolet-visible spectroscopy. This average ratio of protein to DNA was determined to be 1 .1 (0.65) mg protein per ⁇ DNA for three independent batches of protein lysate encapsulated LSNAs.
  • Lysate liposomal SNAs were characterized using transmission electron microscopy, gel electrophoresis, and dynamic light scattering.
  • CryoEM samples were prepared by FEI Vitrobot Mark III by dropping 4 ⁇ _ on a 200- mesh copper TEM grid with lacey carbon film, blotted for 5 seconds, and then plunged into liquid ethane before transferred to a cryo holder and stored in liquid nitrogen prior to imaging.
  • CryoEM imaging was performed using Hitachi HT7700 transmission electron microscope with Gatan cryo-transfer holder under 120kV accelerating voltage, and images were taken with a Gatan imaging camera at a magnification of 30000x.
  • SNAs were prepared by encapsulating AlexaFluor488-labeled lysate and functionalizing cy5-labeled DNA using the same methods described above. Fluorescently labeled SNAs were used to measure the uptake of these particles in mouse macrophages (RAW blue) and mouse bone marrow-derived dendritic cells (BMDCs). RAW blue macrophages were plated at 20,000 cells per well in 24-well plates in DMEM supplemented with 10% fetal bovine serum, 100 ⁇ g/mL normocin, and 200 ⁇ g/mL zeocin.
  • RAW blue macrophages were plated at 20,000 cells per well in 24-well plates in DMEM supplemented with 10% fetal bovine serum, 100 ⁇ g/mL normocin, and 200 ⁇ g/mL zeocin.
  • Bone marrow was isolated from femurs of C57BL/6 mice and cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 1 % penicillin-streptomycin, and 20 ng/mL GM-CSF for six days.
  • BMDCs were added to 24-well plates at 500,000 cells per well, and immediately treated with samples for 1 hour, washed, fixed with 4% paraformaldehyde, and resuspended in phosphate buffered saline for flow cytometry (BD LSRFortessa).
  • BMDCs were cultured as described above and 500,000 cells were treated with samples at 100 nM DNA for 24 hours. Cells and the supernatant were collected for analysis. Cells were incubated with antibodies for CD40 (PE) and CD80 (Brilliant Violet 421 ) for 30 minutes at 4oC, washed and resuspended in phosphate buffered saline for flow cytometry (BD LSRFortessa). The supernatants were used to measure the concentration of cytokines (TNFa, IL-12) using Luminex.
  • PE CD40
  • CD80 Brown Violet 421
  • Samples were then filtered through 70 ⁇ nylon strainers and washed with phosphate buffered saline.
  • the cells were then plated in 10-cm tissue culture plates in RPMI media supplemented with 10% heat-inactivated fetal bovine serum, 1 % penicillin-streptomycin, and 60 ⁇ thioguanine for 10 days.
  • the remaining cell colonies were fixed with methanol and stained with 0.03% methylene blue and then counted.
  • Protein lysates are generated using 4T1 cells, a mouse TNBC cell line, by subjecting these cells to repeated freeze-thaw cycles to cause necrotic cell death and continued until no live cells are remaining. The cellular debris is then purified away from the soluble lysate mixture using centrifugation. The protein content is characterized using SDS-Page electrophoresis and the total protein concentration is measuring using the bicinchoninic acid (BCA) assay.
  • BCA bicinchoninic acid
  • TNBC lysates containing antigens Figure 51
  • Cholesterol terminated DNA was then adsorbed into the lipid bilayer to form liposomal SNAs ( Figure 52).
  • the LSNAs were 73.4 (2.6) nm in diameter with 1 -10 proteins/SNA.
  • the protein lysate is encapsulated within DOPC liposomes using the thin-layer rehydration method.
  • a 1 mg/mL protein solution in phosphate buffered saline is used to rehydrate 5 mM DOPC lipids.
  • Liposomes are formed by five freeze-thaw cycles and 10 rounds of high-pressure extrusion through polycarbonate filters with pore sizes of 200, 100, 80, and 50 nm. Tangential flow filtration with a pore size of 500 kDa is used to remove any non-encapsulated proteins and the sample is repeatedly washed until no protein can be detected in the flow-through.
  • the amount of protein encapsulated within the liposomes is measured using the BCA assay after disruption of the liposome to release encapsulated protein.
  • DNA is functionalized to the liposome surface through hydrophobic interactions between a cholesterol end group and the lipid bilayer.
  • the average ratio of protein to DNA is 1 .1 (0.65) mg protein per umol DNA for three independent batches of protein lysate encapsulated LSNAs.
  • Encapsulation of protein lysates into LSNAs enables higher cellular uptake of lysates into macrophages and dendritic cells compared with lysates encapsulated within bare liposome. Co-delivery of the lysates and DNA into the same cell is increased to 99% of cells compared with when the two components are simply mixed where approximately 70% of cells take up both protein and DNA. Protein lysate LSNAs can activate mouse dendritic cells through toll-like receptor 9 signaling from the DNA, and also through delivery of the lysate.
  • Protein lysates are known to activate dendritic cells through proteins that are released during necrotic cell death that are meant to signal to surrounding cells that there is danger in the area that caused this type of death.
  • Activated dendritic cells increase their expression of co-stimulatory molecules (CD80, CD40, CD86, MHCII) and secretion of pro-inflammatory cytokines (TNFa, IL-12).
  • Vaccination of mice bearing mammary gland 4T1 -tumors with protein lysate LSNAs results in slower tumor growth and a large reduction in lung metastasis.
  • BCA Bicinchoninic Acid
  • the LSNAs allowed for high cellular uptake of TNBC lysate or CpG DNA into antigen presenting cells, including macrophages and dendritic cells, after one hour incubation time in vitro. See Figure 53. Lysate was labeled with Alexa fluor 488 Protein Labeling Kit (Thermo Fisher), encapsulated in liposomes. DNA was Cy-5 labeled. The confocal images represent mouse macrophages treated for 1 hour with 1 uM DNA.
  • FIG. 55 A schematic showing the activation of dendritic cells (DCs) is shown in Figure 55.
  • BMDCs bone marrow derived dendritic cells
  • CpG LSNAs CpG LSNAs
  • Delivered lysate also activated BMDCs without CpG DNA (Figure 56).
  • Expression of co- stimulatory molecules was determined by flow cytometry by staining with fluorescent antibodies. In the experiment, 500,000 dendritic cells were treated for 24 hours with 100 nM DNA.
  • Cytokines were measured with Luminex after the same treatment.
  • 4T1 mouse breast cancer cells were implanted into the mammary gland of Balb/C mice (see Figure 57 for protocol). 4T1 tumors were created on day 0. SNAs were injected on day 6, 1 1 after tumor implantation. Tumor volume was measured by calipers up to 28 days. The control LSNA is lysate encapsulated with non-CpG DNA on the outside
  • TNBC LSNA Vaccine Reduces Lung Metastasis
  • a spherical nucleic acid TNBC vaccine was developed by combining CpG DNA and cell lysates to activate the immune system against TNBC specific antigens.
  • the TNBC LSNA vaccine is readily taken up by immune cells and activates antigen presenting cells in vitro. Treatment of the 4T1 breast cancer model with the TNBC LSNA vaccine resulted in slower growth of the primary tumor and a reduction in lung metastasis.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Mycology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Dispersion Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Vascular Medicine (AREA)
  • Urology & Nephrology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)

Abstract

La présente invention concerne des particules liposomales, des particules liposomales ayant des agents encapsulés, leurs procédés de fabrication et leurs utilisations. Les particules liposomales sont utiles dans la régulation génique et l'administration de médicament.
PCT/US2017/052318 2016-09-19 2017-09-19 Effets thérapeutiques de l'administration cellulaire de petites molécules et de macromolécules avec des acides nucléiques sphériques liposomaux Ceased WO2018067302A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662396733P 2016-09-19 2016-09-19
US62/396,733 2016-09-19

Publications (2)

Publication Number Publication Date
WO2018067302A2 true WO2018067302A2 (fr) 2018-04-12
WO2018067302A3 WO2018067302A3 (fr) 2018-06-07

Family

ID=61831903

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2017/052318 Ceased WO2018067302A2 (fr) 2016-09-19 2017-09-19 Effets thérapeutiques de l'administration cellulaire de petites molécules et de macromolécules avec des acides nucléiques sphériques liposomaux

Country Status (1)

Country Link
WO (1) WO2018067302A2 (fr)

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020168005A1 (fr) * 2019-02-12 2020-08-20 Exicure, Inc. Acide nucléique sphérique combiné et inhibiteur de point de contrôle pour un traitement antitumoral
WO2020181144A1 (fr) * 2019-03-06 2020-09-10 Northwestern University Acide nucléique sphérique conjugué à un oligonucléotide de type épingle à cheveux
WO2020219985A1 (fr) * 2019-04-26 2020-10-29 Exicure, Inc. Administration d'acides nucléiques sphériques pour utilisations ophtalmologiques
WO2021102058A1 (fr) * 2019-11-18 2021-05-27 Children's Medical Center Corporation Stimuli hyperactivant des cellules dendritiques résidentes pour l'immunothérapie anticancéreuse
CN112930184A (zh) * 2018-12-17 2021-06-08 卫材R&D管理有限公司 包含脂质体的配制品
CN114404593A (zh) * 2022-02-22 2022-04-29 武汉叶风生物科技有限公司 一种治疗垂体腺瘤的药物、制备方法及其应用
US11351250B2 (en) 2015-01-12 2022-06-07 Children's Medical Center Corporation Pro-inflammatory and anti-cancer functions of toll-like receptor 4 antagonists
US11364304B2 (en) 2016-08-25 2022-06-21 Northwestern University Crosslinked micellar spherical nucleic acids
WO2022155149A1 (fr) * 2021-01-12 2022-07-21 Northwestern University Acides nucléiques sphériques de nanoparticules lipidiques
US11433131B2 (en) 2017-05-11 2022-09-06 Northwestern University Adoptive cell therapy using spherical nucleic acids (SNAs)
WO2022192038A1 (fr) * 2021-03-12 2022-09-15 Northwestern University Vaccins antiviraux utilisant des acides nucléiques sphériques
US11690920B2 (en) 2017-07-13 2023-07-04 Northwestern University General and direct method for preparing oligonucleotide-functionalized metal-organic framework nanoparticles
US11696954B2 (en) 2017-04-28 2023-07-11 Exicure Operating Company Synthesis of spherical nucleic acids using lipophilic moieties
US11883535B2 (en) 2013-12-03 2024-01-30 Northwestern University Liposomal particles, methods of making same and uses thereof
US12319711B2 (en) 2019-09-20 2025-06-03 Northwestern University Spherical nucleic acids with tailored and active protein coronae
US12378560B2 (en) 2019-10-29 2025-08-05 Northwestern University Sequence multiplicity within spherical nucleic acids

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7410975B2 (en) * 2003-06-20 2008-08-12 Coley Pharmaceutical Group, Inc. Small molecule toll-like receptor (TLR) antagonists
WO2006063252A2 (fr) * 2004-12-09 2006-06-15 Alnylam Pharmaceuticals, Inc. Compositions et methodes pour induire une reponse immunitaire chez un mammifere et methodes pour eviter une reponse immunitaire dirigee contre des agents oligonucleotidiques, notamment des arn interferents courts
WO2009091972A2 (fr) * 2008-01-18 2009-07-23 Alnylam Pharmaceuticals, Inc. Oligonucléotides modifiés chimiquement et petites molécules à utiliser dans la réduction des taux d'activité de microarn et leurs utilisations
WO2010124226A2 (fr) * 2009-04-23 2010-10-28 University Of Massachusetts Biocapteurs sur le récepteur de type toll
WO2015126502A2 (fr) * 2013-12-03 2015-08-27 Northwestern University Particules liposomales, leurs procédés de fabrication et leurs utilisations

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11883535B2 (en) 2013-12-03 2024-01-30 Northwestern University Liposomal particles, methods of making same and uses thereof
US11400153B2 (en) 2015-01-12 2022-08-02 Children's Medical Center Corporation Pro-inflammatory and adjuvant functions of toll-like receptor 4 antagonists
US12070499B2 (en) 2015-01-12 2024-08-27 Children's Medical Center Corporation Pro-inflammatory and adjuvant functions of toll-like receptor 4 antagonists
US12053522B2 (en) 2015-01-12 2024-08-06 Children's Medical Center Corporation Pro-inflammatory and anti-cancer functions of toll-like receptor 4 antagonists
US11351250B2 (en) 2015-01-12 2022-06-07 Children's Medical Center Corporation Pro-inflammatory and anti-cancer functions of toll-like receptor 4 antagonists
US11364304B2 (en) 2016-08-25 2022-06-21 Northwestern University Crosslinked micellar spherical nucleic acids
US11696954B2 (en) 2017-04-28 2023-07-11 Exicure Operating Company Synthesis of spherical nucleic acids using lipophilic moieties
US11433131B2 (en) 2017-05-11 2022-09-06 Northwestern University Adoptive cell therapy using spherical nucleic acids (SNAs)
US11690920B2 (en) 2017-07-13 2023-07-04 Northwestern University General and direct method for preparing oligonucleotide-functionalized metal-organic framework nanoparticles
CN112930184B (zh) * 2018-12-17 2024-07-12 卫材R&D管理有限公司 包含脂质体的配制品
JP7487187B2 (ja) 2018-12-17 2024-05-20 エーザイ・アール・アンド・ディー・マネジメント株式会社 リポソームを含む製剤
EP3897665A4 (fr) * 2018-12-17 2022-09-28 Eisai R&D Management Co., Ltd. Formulation comprenant des liposomes
JP2022515006A (ja) * 2018-12-17 2022-02-17 エーザイ・アール・アンド・ディー・マネジメント株式会社 リポソームを含む製剤
CN112930184A (zh) * 2018-12-17 2021-06-08 卫材R&D管理有限公司 包含脂质体的配制品
WO2020168005A1 (fr) * 2019-02-12 2020-08-20 Exicure, Inc. Acide nucléique sphérique combiné et inhibiteur de point de contrôle pour un traitement antitumoral
US12702719B2 (en) 2019-03-06 2026-08-11 Northwestern University Hairpin-like oligonucleotide-conjugated spherical nucleic acid
WO2020181144A1 (fr) * 2019-03-06 2020-09-10 Northwestern University Acide nucléique sphérique conjugué à un oligonucléotide de type épingle à cheveux
WO2020219985A1 (fr) * 2019-04-26 2020-10-29 Exicure, Inc. Administration d'acides nucléiques sphériques pour utilisations ophtalmologiques
US12319711B2 (en) 2019-09-20 2025-06-03 Northwestern University Spherical nucleic acids with tailored and active protein coronae
US12378560B2 (en) 2019-10-29 2025-08-05 Northwestern University Sequence multiplicity within spherical nucleic acids
US20230076515A1 (en) * 2019-11-18 2023-03-09 Children's Medical Center Corporation Stimuli that hyperactivate resident dendritic cells for cancer immunotherapy
IL293030B1 (en) * 2019-11-18 2025-12-01 Childrens Medical Center Stimuli that hyperactivate resident dendritic cells for cancer immunotherapy
WO2021102058A1 (fr) * 2019-11-18 2021-05-27 Children's Medical Center Corporation Stimuli hyperactivant des cellules dendritiques résidentes pour l'immunothérapie anticancéreuse
CN114980919A (zh) * 2019-11-18 2022-08-30 儿童医学中心公司 过度活化驻留树突状细胞的刺激物用于癌症免疫疗法
EP4061409A4 (fr) * 2019-11-18 2023-12-27 Children's Medical Center Corporation Stimuli hyperactivant des cellules dendritiques résidentes pour l'immunothérapie anticancéreuse
CN114980919B (zh) * 2019-11-18 2026-04-14 儿童医学中心公司 过度活化驻留树突状细胞的刺激物用于癌症免疫疗法
JP2023502400A (ja) * 2019-11-18 2023-01-24 ザ・チルドレンズ・メデイカル・センター・コーポレーシヨン がん免疫療法のために常在型樹状細胞を超活性化する刺激
IL293030B2 (en) * 2019-11-18 2026-04-01 Childrens Medical Center Stimuli that cause hyperactivity of local dendritic cells for cancer immunotherapy
JP7813226B2 (ja) 2019-11-18 2026-02-12 ザ・チルドレンズ・メデイカル・センター・コーポレーシヨン がん免疫療法のために常在型樹状細胞を超活性化する刺激
CN116940324A (zh) * 2021-01-12 2023-10-24 西北大学 脂质纳米颗粒球形核酸
WO2022155149A1 (fr) * 2021-01-12 2022-07-21 Northwestern University Acides nucléiques sphériques de nanoparticules lipidiques
WO2022192038A1 (fr) * 2021-03-12 2022-09-15 Northwestern University Vaccins antiviraux utilisant des acides nucléiques sphériques
US12691166B2 (en) 2021-03-12 2026-07-28 Northwestern University Antiviral vaccines using spherical nucleic acids
CN114404593A (zh) * 2022-02-22 2022-04-29 武汉叶风生物科技有限公司 一种治疗垂体腺瘤的药物、制备方法及其应用
CN114404593B (zh) * 2022-02-22 2023-10-27 武汉叶风生物科技有限公司 一种治疗垂体腺瘤的药物、制备方法及其应用

Also Published As

Publication number Publication date
WO2018067302A3 (fr) 2018-06-07

Similar Documents

Publication Publication Date Title
US20260060927A1 (en) Liposomal particles, methods of making same and uses thereof
JP7600208B2 (ja) 拘束された脂質を含むナノ材料およびその使用
CN113645960B (zh) 含有氧化的胆固醇的药物递送系统
EP2377517B1 (fr) Composition pharmaceutique contenant un médicament anionique et procédé de préparation associé
US20230147733A1 (en) Oxidized tumor cell lysates encapsulated in liposomal spherical nucleic acids as potent cancer immunotherapeutics
WO2018209270A1 (fr) Thérapie cellulaire adoptive utilisant des acides nucléiques sphériques (sna)
US20240165263A1 (en) Targeting multiple t cell types using spherical nucleic acid vaccine architecture
KR20240102993A (ko) 암의 면역요법 치료를 위한 cGAS-STING 및 STAT3 경로 조절용 구형 핵산

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17858888

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17858888

Country of ref document: EP

Kind code of ref document: A2