WO2019204451A1 - Apport de médicament sous forme de nanoparticules lipidiques améliorées, à l'aide d'un polymère chargé négativement - Google Patents

Apport de médicament sous forme de nanoparticules lipidiques améliorées, à l'aide d'un polymère chargé négativement Download PDF

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WO2019204451A1
WO2019204451A1 PCT/US2019/027885 US2019027885W WO2019204451A1 WO 2019204451 A1 WO2019204451 A1 WO 2019204451A1 US 2019027885 W US2019027885 W US 2019027885W WO 2019204451 A1 WO2019204451 A1 WO 2019204451A1
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cholesterol
lipid
peg
polyanionic
helper
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Kathryn A. WHITEHEAD
Rebecca BALL
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Carnegie Mellon University
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    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • 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
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0031—Rectum, anus
    • 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
    • A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
    • 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
    • A61K9/0043—Nose
    • 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
    • A61K9/0046—Ear
    • 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
    • A61K9/0048—Eye, e.g. artificial tears
    • 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
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • 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
    • A61K9/02—Suppositories; Bougies; Bases therefor; Ovules
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides

Definitions

  • RNA lipid nanoparticle
  • these formulations include a cationic or ionizable lipid, cholesterol or its analogues such as oxidized cholesterol or desmosterol, a“helper” lipid such as DOPC or DOPE, and a lipid conjugated to the polymer polyethylene glycol (PEG).
  • the cationic/ionizable lipid is the active delivery agent. Cholesterol or its analogues and the helper lipid aid in the molecular packing and stability of the particle. PEG reduces the degree to which particles are cleared by immune system in vivo.
  • LNP/siRNA drug product recently approved in the United States is patisiran (ONPATTRO ® , Anylam Pharmaceuticals), an siRNA for knocking down transthyretin, for use in treatment of hereditary transthyretin amyloidosis.
  • patisiran ONPATTRO ® , Anylam Pharmaceuticals
  • siRNA for knocking down transthyretin
  • a variety of other lipid-based delivery systems are undergoing clinical trials for delivery of siRNA, mRNA, and plasmid vectors, antisense reagents, among other nucleic acid or nucleic acid analog therapeutic agents.
  • a lipid particle e.g., a lipid nanoparticle
  • the lipid particle comprises: an ionizable or cationic lipid; cholesterol or a cholesterol analogue; a helper lipid; a polyethyleneglycol-lipid (PEG-lipid) or polyethyleneglycol-cholesterol (PEG-cholesterol) comprising a polyethyleneglycol moiety and a lipid or cholestorol moiety; a polyanionic (having a plurality of negative charges) therapeutic agent; and a polyanionic helper polymer.
  • PEG-lipid polyethyleneglycol-lipid
  • PEG-cholesterol polyethyleneglycol-cholesterol
  • a method of making a lipid particle e.g., a lipid nanoparticle.
  • the method comprises mixing in solution, e.g., an aqueous solution: an ionizable or cationic lipid; cholesterol or a cholesterol analogue; a helper lipid; a polyethyleneglycol-lipid (PEG-lipid) or polyethyleneglycol-cholesterol (PEG- cholesterol) comprising a polyethyleneglycol moiety and a lipid or cholestorol moiety; a polyanionic therapeutic agent; and a polyanionic helper polymer, in amounts effective to produce a lipid particle.
  • an aqueous solution an ionizable or cationic lipid
  • cholesterol or a cholesterol analogue e.g., a helper lipid
  • PEG-lipid polyethyleneglycol-lipid
  • PEG- cholesterol polyethyleneglycol-cholesterol
  • a method of delivering a polyanionic therapeutic agent to a cell or to a patient comprises, administering a lipid particle, e.g., a lipid nanoparticle, to the cell or patient comprising: an ionizable or cationic lipid; cholesterol or a cholesterol analogue; a helper lipid; a polyethyleneglycol-lipid (PEG- lipid) or polyethyleneglycol-cholesterol (PEG-cholesterol) comprising a polyethyleneglycol moiety and a lipid or cholesterol moiety; a polyanionic therapeutic agent; and a polyanionic helper polymer.
  • the therapeutic agent is mRNA or an RNAi agent, such as an siRNA.
  • a lipid particle comprising:
  • helper lipid a polyethyleneglycol-lipid (PEG-lipid) or polyethyleneglycol-cholesterol (PEG- cholesterol) comprising a polyethyleneglycol moiety and a lipid or cholesterol moiety; a polyanionic therapeutic agent; and
  • Clause 2 The particle of clause 1 , wherein the polyanionic therapeutic agent is a nucleic acid or a nucleic acid analog having a negatively-charged backbone.
  • Clause 3 The particle of clause 1 , wherein the polyanionic therapeutic agent is RNA.
  • Clause 4 The particle of clause 1 , wherein the polyanionic therapeutic agent is an mRNA or an RNAi agent.
  • Clause 5 The particle of clause 1 , wherein the polyanionic therapeutic agent is an siRNA.
  • Clause 6 The particle of clause 1 , wherein the polyanionic therapeutic agent is an mRNA.
  • Clause 7 The particle of clause 1 , wherein the polyanionic therapeutic agent is an siRNA, and the polyanionic helper polymer is an mRNA.
  • Clause 8 The particle of clause 1 , wherein the polyanionic therapeutic agent is an mRNA, and the polyanionic helper polymer is an siRNA.
  • Clause 10 The particle of any one of clauses 1-9, wherein the anionic helper polymer has a pKa of less than 2.
  • Clause 11 The particle of any one of clauses 1-10, wherein the anionic helper polymer is a polystyrene having a plurality of acidic pendant groups.
  • Clause 12 The particle of any one of clauses 1-11 , wherein the anionic helper polymer has a hydrocarbon backbone having pendant sulfonate, carboxyl, or phosphinic acid moieties.
  • Clause 13 The particle of any one of clauses 1 -12, wherein the ionizable or cationic lipid is an ionizable amine lipid.
  • Clause 14 The particle of clause 13, wherein the ionizable or cationic lipid is heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3- DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3]-dioxolane (DLin-KC2-DMA), or 3060no.
  • Clause 15 The particle of any one of clauses 1 -14, wherein the helper lipid is DSPC, DSPE, DOPC, or DOPE.
  • Clause 16 The particle of any one of clauses 1 -15, wherein the PEG-lipid or polyethyleneglycol-cholesterol (PEG-cholesterol) comprises a C10-C20 saturated or unsaturated fatty acid and a PEG moiety ranging from 300 g/mol to 5000 g/mol.
  • PEG-cholesterol polyethyleneglycol-cholesterol
  • the PEG-lipid or PEG-cholesterol comprises a PEG-ceramide, a PEG-cholesterol, a PEG-phosphoethanolamine, a DMG-PEG (1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), a DSG- PEG (distearoyl-rac-glycerol-PEG2K), or a DSPE-PEG.
  • Clause 18 The lipid particle of any one of clauses 1 -17, wherein the polyanionic therapeutic agent or the polyanionic helper polymer is a therapeutic mRNA.
  • Clause 19 The lipid particle of any one of clauses 1 -17, wherein the polyanionic therapeutic or the polyanionic helper polymer is a therapeutic siRNA.
  • the weight ratio of ionizable or cationic lipid to the sum of the weights of the polyanionic therapeutic agent and the helper polymer ranges from 7:1 to 10:1 ;
  • the weight ratio of polyanionic helper polymer to the polyanionic therapeutic agent ranges from 0.01 :1 to 1 1 :1 ;
  • the molecular weight of polyanionic helper polymer ranges from 4 - 100 kDa; and the molar ratio of ionizable or cationic lipid to cholesterol or cholesterol analogue to helper lipid to polyethyleneglycol-lipid (PEG-lipid) or polyethyleneglycol-cholesterol (PEG-cholesterol)is 35-55:30-50:0-20:0.5-5.
  • mRNA or from 1 kDa to 200 kDa PSS.
  • Clause 24 The lipid particle of any one of clauses 1 -23, wherein the helper polymer has a polydispersity index of less than 2.
  • helper lipid a helper lipid
  • polyethyleneglycol-lipid PEG-lipid
  • PEG- cholesterol polyethyleneglycol-cholesterol
  • a polyanionic helper polymer in amounts effective to produce a lipid particle.
  • Clause 26 The method of clause 25, wherein the polyanionic therapeutic agent is a nucleic acid or a nucleic acid analog having a negatively-charged backbone.
  • Clause 27 The method of clause 25, wherein the polyanionic therapeutic agent is RNA.
  • Clause 28 The method of clause 25, wherein the polyanionic therapeutic agent is an mRNA or an RNAi agent.
  • Clause 29 The method of clause 25, wherein the polyanionic therapeutic agent is an siRNA.
  • Clause 30 The method of clause 25, wherein the polyanionic therapeutic agent is an mRNA.
  • Clause 31 The method of clause 25, wherein the polyanionic therapeutic agent is an siRNA, and the polyanionic helper polymer is an mRNA.
  • Clause 32 The method of clause 25, wherein the polyanionic therapeutic agent is an mRNA, and the polyanionic helper polymer is an siRNA.
  • Clause 33 The method of any one of clauses 25-32, wherein the anionic helper polymer has a pKa of less than 4.
  • Clause 35 The method of any one of clauses 25-34, wherein the anionic helper polymer is a polystyrene having a plurality of acidic pendant groups.
  • Clause 36 The method of any one of clauses 25-35, wherein the anionic helper polymer has a hydrocarbon backbone having pendant sulfonate, carboxyl, or phosphinic acid moieties.
  • Clause 37 The method of any one of clauses 25-36, wherein the ionizable or cationic lipid is an ionizable amine lipid.
  • the PEG-lipid or PEG-cholesterol comprises PEG-ceramide, a PEG-cholesterol, a PEG-phosphoethanolamine, a DMG- PEG (1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), a DSG-PEG (distearoyl-rac-glycerol-PEG2K), or a DSPE-PEG.
  • the polyanionic therapeutic agent or the polyanionic helper polymer is a therapeutic mRNA.
  • Clause 44 The method of any one of clauses 25-42, wherein the polyanionic therapeutic or the polyanionic helper polymer is a therapeutic siRNA.
  • the weight ratio of ionizable or cationic lipid to the sum of the weights of the polyanionic therapeutic agent and the helper polymer ranges from 7:1 to 10:1 ;
  • the weight ratio of polyanionic helper polymer to the polyanionic therapeutic agent ranges from 0.01 :1 to 1 1 :1 ;
  • the molecular weight of polyanionic helper polymer ranges from 4 - 100 kDa; and the molar ratio of ionizable or cationic lipid to cholesterol or cholesterol analogue to helper lipid to polyethyleneglycol-lipid (PEG-lipid) or PEG-cholesterol is 35-55:30- 50:0-20:0.5-5.
  • the ionizable or cationic lipid is DLin-MC3-DMA, DLin-KC2-DMA, CKK-E12, or 3060no;
  • the helper lipid is DSPC, DSPE, DOPC, or DOPE;
  • the polyanionic therapeutic agent is mRNA or siRNA
  • the polyanionic helper polymer is from 1 kDa to 200 kDa PSS.
  • the ionizable or cationic lipid is 3060no;
  • helper lipid is DSPC
  • polyethyleneglycol-lipid PEG-lipid
  • polyethyleneglycol-cholesterol PEG- cholesterol
  • the polyanionic therapeutic agent is mRNA or siRNA
  • the polyanionic helper polymer is from 1 kDa to 200 kDa PSS.
  • the ionizable or cationic lipid is 3060no;
  • helper lipid is DSPC
  • polyethyleneglycol-lipid PEG-lipid
  • polyethyleneglycol-cholesterol PEG- cholesterol
  • the polyanionic therapeutic agent is siRNA; and the polyanionic helper polymer is mRNA or from 1 kDa to 200 kDa PSS.
  • Clause 49 The method of any one of clauses 25-48, wherein the helper polymer has a polydispersity index of less than 2.
  • Clause 50 A method of delivering a polyanionic therapeutic agent to a cell or to a patient, comprising, administering a lipid particle of any one of clauses 1 -24 to the cell or patient.
  • Clause 51 The method of clause 50, wherein the therapeutic agent is delivered parenterally to a patient.
  • Clause 52 The method of clause 50, wherein the therapeutic agent is delivered orally or rectally to a patient.
  • Clause 53 The method of clause 50-52, wherein the therapeutic agent is an mRNA or an RNAi agent, which is administered in an amount effective to increase or decrease gene expression in the patient.
  • FIG. 1 Lipid nanoparticles co-delivered siRNA and mRNA in vitro.
  • HeLa cells that stably expressed firefly luciferase were incubated with LNPs containing 10 nM of siRNA against firefly luciferase and/or 100 ng of mRNA encoding mCHERRY. Expression of both proteins was assessed 24 hours post-transfection.
  • A) LNPs co-formulated with siRNA and mRNA resulted in greater gene silencing than LNPs formulated only with siRNA. (n 12 - 18)
  • D) The RNA cargo, but not the LNP formulation number, affected LNP size (n 2 - 3).
  • Figure 2 Lipid nanoparticles synergistically co-delivered siRNA and mRNA in mice. All animals received siRNA specific for Factor VII (FVII) at a dose of 0.03 mg/kg and/or mRNA encoding firefly luciferase at a dose of 0.5 mg/kg.
  • FVII Factor VII
  • A) LNPs co-formulated with both RNAs (purple circles) using Formulations 3 - 5 induced higher levels of gene silencing than LNPs formulated with only siRNA (blue triangle) and only mRNA (control, red square) (n 3)
  • mice received an injection containing LNPs formulated with siFVII combined with LNPs formulated with mLuc.
  • FIG. 3 A) LNP formulation 4 resulted in the highest luciferase expression in mice. The vast majority of signal was produced in the liver and spleen. IVIS images were taken 6 hours after tail vein injection of LNPs with 0.5 mg/kg mRNA encoding firefly luciferase (mLUC) and/or 0.03 mg/kg siRNA against FVII (siFVII).
  • mLUC firefly luciferase
  • siFVII siRNA against FVII
  • Figure 4 Delivery synergy from co-formulation depended on the total RNA concentration.
  • A) The addition of“helper” mRNA to an LNP intended to deliver siRNA improved gene silencing efficacy for intermediate total RNA concentrations. All mice were dosed with 0.03 mg/kg siRNA specific for Factor VII (FVII). (n 3)
  • LNP efficacy depended on the total RNA and lipidoid concentrations.
  • Figure 5 The addition of“helper” siRNA to LNPs loaded with luciferase encoding mRNA improved mRNA delivery for the siRNA: mRNA weight ratio of 0.006 : 0.1 .
  • Figure 6 For A, C, & E LNPs were formulated using Formulation 4 with a constant siRNA concentration (0.006 mg/ml_) and varied mRNA concentrations (0.05-0.15 mg/ml_).
  • mice were dosed with 0.03 mg/kg siFVII.
  • B, D, & F LNPs were formulated using Formulation 4 with a constant mRNA concentration (0.1 mg/mL) and varied siRNA concentration (0 - 0.08 mg/mL). All mice were dosed with 0.5 mg/kg mLuc.
  • A, B) LNPs did not induce weight change compared to untreated mice (n 3)
  • C, D) RNA entrapment in LNPs changed with total RNA concentration during formulation (n 3 technical replicates)
  • FIG. 8 LNPs were formulated with siFVII alone, siFVII + mLuc (0.006 mg/mL) or LNPs with siFVII + mLuc (0.1 mg/mL) while keeping the lipidoid concentration constant. LNPs were delivered to mice at 0.03 mg/kg siFVII.
  • A) LNPs did not form properly when formulated with 0.006 mg/mL siRNA, 0.1 mg/mL mLuc and a low concentration of lipidoid (0.05 mg/mL). (n 3 technical replicates)
  • B) The z- average diameter was measured using dynamic light scattering (n 3 technical replicates).
  • C) LNPs did not induce weight loss in mice compared to an untreated group (n 3) In all panels, error bars represent s.d.
  • Figure 9 Inclusion of a negatively charged“helper” polymer in single RNA LNP formulations improved efficacy.
  • LNPs formulated with helper mRNA, 70 kDa PSS, or PolyU improved knockdown compared to LNPs formulated without helper polymer (n 3 - 4)
  • LNPs formulated with PSS facilitated comparable FVII silencing to LNPs without PSS using three times less drug.
  • PSS-formulated LNPs induced dose-dependent gene knockdown (n 3).
  • Figure 10 The addition of siFVII or PSS to the LNP formulation significantly enhanced mLuc delivery.
  • FIG. 12 LNPs loaded with siFVII were formulated with or without PSS and delivered to mice at a range of siFVII doses (0.005 - 0.03 mg/kg).
  • A) RNA entrapment decreased with decreasing siFVII dose (n 3 technical replicates)
  • B) The z-average diameter was measured for each LNP using dynamic light scattering (n 3 technical replicates)
  • C) Mice treated with LNPs did not experience weight change two days post-injection compared to untreated animals (n 3) In all panels, error bars represent s.d.
  • the terms“comprising,”“comprise” or“comprised,” and variations thereof, in reference to elements of an item, composition, apparatus, method, process, system, claim etc. are intended to be open-ended, meaning that the item, composition, apparatus, method, process, system, claim etc. includes those elements and other elements can be included and still fall within the scope/definition of the described item, composition, apparatus, method, process, system, claim etc.
  • "a” or “an” means one or more.
  • “another” may mean at least a second or more.
  • patient or “subject” refer to members of the animal kingdom, including, but not limited to human beings.
  • a composition is "biocompatible" in that the composition and, where applicable, degradation products thereof, are substantially non-toxic to cells or organisms within acceptable tolerances, including substantially non-carcinogenic and substantially non- immunogenic, and are cleared or otherwise degraded in a biological system, such as an organism (patient) without substantial toxic effect.
  • degradation mechanisms within a biological system include chemical reactions, hydrolysis reactions, and enzymatic cleavage.
  • polymer composition is a composition comprising one or more polymers.
  • polymers includes, without limitation, homopolymers, heteropolymers, co-polymers, or block co-polymers and can be both natural and/or synthetic. Homopolymers contain one type of building block, or monomer, whereas copolymers contain more than one type of monomer.
  • (co)polymer” and like terms refer to either homopolymers or copolymers.
  • Polymers can be linear, or have branched structures, such as comb, star, or dendrimeric structures. Polymers may be cross-linked.
  • a polymer comprises a backbone, and often comprises a pendant group, such as an anionic or cationic moiety, or a reactive group.
  • a polymer “comprises” or is “derived from” a stated monomer if that monomer is incorporated into the polymer.
  • the incorporated monomer (monomer residue) that the polymer comprises is not the same as the monomer prior to incorporation into a polymer, in that at the very least, certain groups are missing and/or modified when incorporated into the polymer backbone.
  • a polymer is said to comprise a specific type of linkage if that linkage is present in the polymer.
  • a dosage form e.g., an oral dosage form, a topical dosage form, or a parenteral dosage form, e.g., an intravenous or intramuscular dosage form, for delivery of polyanions, such as nucleic acids or nucleic acid analogs to a patient.
  • the polyanions are single stranded or double stranded nucleic acids or nucleic acid analogs, included single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, or modified versions of any of the preceding, or analogs of any of the preceding.
  • the nucleic acid or nucleic acid analog may be, without limitation: mRNA, siRNA, miRNA (microRNA), gRNA (guide RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), tmRNA (transfer-messenger RNA), piRNA (Piwi-interacting RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), scaRNAs (small Cajal body RNA), Y RNA, eRNA (enhancer RNA), shRNA (small hairpin RNA), stRNA (small temporal RNA), DNA, chloroplast DNA, cDNA (complementary DNA), gDNA (genomic DNA), Hachimoji DNA, mitochondrial DNA, msDNA (multicopy single-stranded DNA), XNA (xeno nucleic acid), glycol nucleic acid, threose nucleic acid, he
  • the dosage form comprises a lipid particle, such as a lipid nanoparticle, comprising the polyanionic therapeutic agent and a polyanionic polymer different from the nucleic acid or nucleic acid analog, and in aspects is not a nucleic acid or nucleic acid analog.
  • compositions are prepared in accordance with acceptable pharmaceutical procedures, such as described in Remington: The Science and Practice of Pharmacy. 21 st edition, ed. Paul Beringer et ai, Lippincott, Williams & Wilkins, Baltimore, MD Easton, Pa. (2005) (see, e.g., Chapters 37, 39, 41 , 42 and 45 for examples of powder, liquid, parenteral, intravenous and oral solid formulations and methods of making such formulations).
  • the dosage form may comprise additional carriers or excipients, such as water, saline (e.g., normal saline), or phosphate-buffered saline, as are broadly-known in the pharmaceutical arts.
  • compositions may comprise a pharmaceutically acceptable carrier, or excipient.
  • An excipient is an inactive substance used as a carrier for the active ingredients of a medication. Although“inactive,” excipients may facilitate and aid in increasing the delivery, stability or bioavailability of an active ingredient in a drug product.
  • Non-limiting examples of useful excipients include: anti-adherents, binders, rheology modifiers, coatings, disintegrants, emulsifiers, oils, buffers, salts, acids, bases, fillers, diluents, solvents, flavors, colorants, glidants, lubricants, preservatives, antioxidants, sorbents, vitamins, sweeteners, etc., as are available in the pharmaceutical/compounding arts.
  • the dosage form may comprise a delayed-release coating, such as an enteric coating surrounding the LNPs comprising the therapeutic agent to delay release of the therapeutic agent until it reaches to small intestine.
  • the dosage form may be provided as a unit dosage form, e.g., with lipid particles packaged within a syringe or ampoule for single or multiple use.
  • the dosage form also may be topical, e.g., for delivery to a body surface, such as skin, mucosa, or other topical routes, including, dermal, oral, nasal, optic, otic, or vaginal delivery routes.
  • the dosage form also may be a suitable gastrointestinal dosage form, for example as a suppository, or through a feeding tube.
  • polyanions such as nucleic acids or nucleic acid analogs
  • the polyanions include single stranded or double stranded nucleic acids or nucleic acid analogs, including single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, or modified versions of any of the preceding, or analogs of any of the preceding.
  • the nucleic acid or nucleic acid analog may be, without limitation: mRNA, siRNA, miRNA (microRNA), gRNA (guide RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), tmRNA (transfer-messenger RNA), piRNA (Piwi-interacting RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), scaRNAs (small Cajal body RNA), Y RNA, eRNA (enhancer RNA), shRNA (small hairpin RNA), stRNA (small temporal RNA), DNA, chloroplast DNA, cDNA (complementary DNA), gDNA (genomic DNA), Hachimoji DNA, mitochondrial DNA, msDNA (multicopy single-stranded DNA), XNA (xeno nucleic acid), glycol nucleic acid, threose nucleic acid, he
  • an “effective amount” or “amount effective” to achieve a desirable therapeutic, pharmacological, medicinal, or physiological effect is any amount that achieves the stated purpose.
  • an effective amount is a bioactive amount of a therapeutic agent delivered to the patient, or an organ or tissue of the patient. Based on the teachings provided herein, one of ordinary skill can readily ascertain effective amounts of the elements of the described dosage form and produce a safe and effective dosage form and drug product.
  • a therapeutic agent is any compound or composition that is delivered to a patient to achieve a desired effect, such as a beneficial, treatment, or curative effect.
  • Therapeutic agents include proteins, such as polypeptides or proteins.
  • therapeutic agents are polyanions, such as single stranded or double stranded nucleic acids or nucleic acid analogs having a negatively-charged backbone, including, without limitation single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, or modified versions of any of the preceding, or analogs of any of the preceding.
  • the nucleic acid or nucleic acid analog may be, without limitation: mRNA, siRNA, miRNA (microRNA), gRNA (guide RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), tmRNA (transfer-messenger RNA), piRNA (Piwi-interacting RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), scaRNAs (small Cajal body RNA), Y RNA, eRNA (enhancer RNA), shRNA (small hairpin RNA), stRNA (small temporal RNA), DNA, chloroplast DNA, cDNA (complementary DNA), gDNA (genomic DNA), Hachimoji DNA, mitochondrial DNA, msDNA (multicopy single-stranded DNA), XNA (xeno nucleic acid), glycol nucleic acid, threose nucleic acid, he
  • Nucleic acids and nucleic acid analogs comprise a backbone and a sequence of nucleobases.
  • the backbone monomer residues can be any suitable nucleic acid backbone monomer residues having a negative charge, such as a ribose or deoxyribose connected by a phosphodiester bond, or a backbone residue of a nucleic acid analog monomer.
  • the backbone monomer includes both the structural“residue” component, such as the ribose in RNA, and any active groups that are modified in linking monomers together, such as the 5’ triphosphate and 3’ hydroxyl groups of a ribonucleotide, which are modified when polymerized into RNA to leave a negatively-charged phosphodiester linkage.
  • structural“residue” component such as the ribose in RNA
  • any active groups that are modified in linking monomers together such as the 5’ triphosphate and 3’ hydroxyl groups of a ribonucleotide, which are modified when polymerized into RNA to leave a negatively-charged phosphodiester linkage.
  • Non-limiting examples of common nucleic acid analogs include peptide nucleic acids, such as phosphorothioate nucleic acids, locked nucleic acid (2’-0-4’-C- methylene bridge, including oxy, thio or amino versions thereof), unlocked nucleic acid (the C2’-C3’ bond is cleaved), 2’-0-methyl-substituted RNA, morpholino nucleic acid, threose nucleic acid, and glycol nucleic acid, among others, as are broadly-known.
  • peptide nucleic acids such as phosphorothioate nucleic acids, locked nucleic acid (2’-0-4’-C- methylene bridge, including oxy, thio or amino versions thereof), unlocked nucleic acid (the C2’-C3’ bond is cleaved), 2’-0-methyl-substituted RNA, morpholino nucleic acid, threose nucleic acid, and glycol
  • Nucleobases are recognition moieties that bind specifically to one or more of adenine, guanine, thymine, cytosine, and uracil, e.g., by Watson-Crick or Watson-Crick-like base pairing by hydrogen bonding.
  • A“nucleobase” includes primary nucleobases: adenine, guanine, thymine, cytosine, and uracil, as well as modified purine and pyrimidine bases, such as, without limitation, hypoxanthine, xanthene, 7- methylguanine, 5, 6, di hydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine (see also, e.g., U.S.
  • nucleobases are known in the art, for example as disclosed in United States Patent Nos. 8,053,212, 8,389,703, and 8,653,254. Nucleobases may be divalent, e.g., as in International Patent Publication Nos. WO 2014/169206 and WO 2018/058091 . Suitable nucleic acids, or modified nucleic acids, such as nucleic acid analogs are broadly-known to those of skill in the art.
  • A“gene” is a sequence of DNA or RNA which codes for a molecule, such as a protein or a functional RNA, such as a ncRNA that has a function.
  • Complementary refers to the ability of polynucleotides (nucleic acids) to hybridize to one another, forming inter-strand base pairs. Base pairs are formed by hydrogen bonding between nucleotide units in antiparallel polynucleotide strands.
  • Complementary polynucleotide strands can base pair (hybridize) in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes.
  • Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product, e.g., a protein or functional RNA. Gene expression involves various steps, including transcription, translation, and post- translational modification of a protein.
  • Transcription is the process by which the DNA gene sequence is transcribed into pre-mRNA (messenger RNA).
  • the steps include: RNA polymerase, together with one or more general transcription factors, binds to promoter DNA.
  • Transcription factors are proteins that control the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence (i.e., the promoter region). The function of TFs is to regulate genes in order to make sure that they are expressed in the right cell at the right time and in the right amount throughout the life of the cell and the organism.
  • the promoter region of a gene is a region of DNA that initiates transcription of that particular gene.
  • Promoters are located near the transcription start sites of genes, on the same strand, and often, but not exclusively, are upstream (towards the 5' region of the sense strand) on the DNA. Promoters can be about 100-1000 base pairs long. Additional sequences and noncoding elements can affect transcription rates.
  • the RNA is further processed. This includes polyadenylation, capping, and splicing. Polyadenylation is the addition of a poly(A) tail to a messenger RNA.
  • the poly(A) tail consists of a chain of adenosine monophosphates. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation.
  • Capping refers to the process wherein the 5’ end of the pre-mRNA has a specially altered nucleotide.
  • the 5’ cap (cap-0) found on the 5’ end of an mRNA molecule, consists of a guanine nucleotide connected to mRNA via an unusual 5’ to 5’ triphosphate linkage.
  • pre-mRNA is edited. Specifically, during this process, introns are removed, and exons are joined together. The resultant product is known as mature mRNA.
  • the RNA may remain in the nucleus or exit to the cytoplasm through the nuclear pore complex.
  • mRNA encoding a protein can be delivered to cells by the lipid particles, such as lipid nanoparticles, and methods described herein to produce the protein in the cells.
  • the protein may be therapeutic.
  • mRNA can be produced by in vitro transcription (IVT) as is broadly-known (see, e.g., Kwon, et al. Emergence of synthetic mRNA: In vitro synthesis of mRNA and its applications in regenerative medicine. Biomaterials 156; February 2018:172-193, describing the design and preparation of IVT mRNA, chemical modification of IVT mRNA, and therapeutic applications of IVT mRNA in regenerative medicine, cellular reprogramming, stem cell engineering, and protein replacement therapy).
  • IVT in vitro transcription
  • RNA levels in a cell can be controlled post- transcriptionally.
  • Native mechanisms for controlling RNA levels include endogenous gene silencing mechanisms, interference with translational mechanisms, interference with RNA splicing mechanisms, and destruction of duplexed RNA by RNAse H, or RNAse H-like activity. As is broadly-recognized by those of ordinary skill in the art, these endogenous mechanisms can be exploited to decrease or silence mRNA activity in a cell or organism in a sequence-specific, targeted manner.
  • Antisense technology typically involves administration of a single-stranded antisense oligonucleotide (ASO) that is chemically-modified, e.g., as described herein, for bio-stability, and is administered in sufficient amounts to effectively penetrate the cell and bind in sufficient quantities to target mRNAs in cells.
  • RNA interference harnesses an endogenous and catalytic gene silencing mechanism, which means that once, e.g., a microRNA, or double-stranded siRNA has been delivered into the cytosol, they are efficiently recognized and stably incorporated into the RNA-induced silencing complex (RiSC) to achieve prolonged gene silencing.
  • RNAi agent refers to an agent that contains RNA nucleotides, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).
  • RNAi RNA interference
  • the iRNA modulates, e.g., knocks down or silences, the expression of an RNA in a cell, e.g., a cell within a subject, such as a mammalian subject.
  • an RNAi agent includes a single stranded RNAi that interacts with a target RNA sequence to direct the cleavage of the target RNA.
  • siRNAs double stranded short interfering RNAs
  • Dicer a Type III endonuclease known as Dicer.
  • Dicer a ribonuclease-lll-like enzyme, processes these dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs.
  • siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition.
  • RISC RNA-induced silencing complex
  • one or more endonucleases within the RISC cleave the target to induce silencing.
  • the invention relates to a single stranded RNA (ssRNA) (the antisense strand of an siRNA duplex) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene.
  • siRNA is also used herein to refer to an interfering RNA (iRNA).
  • the RNAi agent may be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA.
  • Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA.
  • the single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are described in U.S. Patent No. 8,101 ,348 and in Lima et al., (2012) Cell 150:883-894. Any of the RNAi agents described herein may be used as a single- stranded siRNA as described herein or as chemically modified by the methods described in Lima et al.
  • an "iRNA” or RNAi agent” for use in the compositions and methods described herein is a double stranded RNA and can be referred to herein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”.
  • dsRNA refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having "sense” and “antisense” orientations with respect to a target RNA.
  • a double stranded RNA triggers the degradation of a target RNA, e.g., an mRNA, through a post- transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.
  • each strand of a dsRNA molecule may be ribonucleotides, but as described in detail herein, each or both strands can also include nucleotide analogs, where one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide.
  • an "RNAi agent” or“RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides.
  • modified nucleotide refers to a nucleotide having, independently, a modified sugar moiety, a modified inter-nucleotide linkage, and/or modified nucleobase.
  • modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to inter-nucleoside linkages, sugar moieties, or nucleobases.
  • the modifications suitable for use in the agents described herein include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by "RNAi agent” or“RNAi reagent” for the purposes of this disclosure.
  • the duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21 , 15-20, 15- 19, 15-18, 15- 17, 18-30, 18-29, 18-28,
  • the two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a "hairpin loop.”
  • a hairpin loop can comprise at least one unpaired nucleotide. In some aspects, the hairpin loop can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides.
  • the hairpin loop can be 10 or fewer nucleotides. In some aspects, the hairpin loop can be 8 or fewer unpaired nucleotides. In some aspects, the hairpin loop can be 4-10 unpaired nucleotides. In some aspects, the hairpin loop can be 4-8 nucleotides.
  • RNA molecules are comprised by separate RNA molecules, those molecules need not, but can be covalently connected.
  • the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a "linker.”
  • the RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex.
  • an RNAi agent may comprise one or more nucleotide overhangs.
  • an RNAi agent is a dsRNA, each strand of which comprises 19-23 nucleotides, that interacts with a target RNA sequence without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer.
  • Dicer a ribonuclease-lll-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs.
  • the siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition.
  • RISC RNA-induced silencing complex
  • an RNAi agent is a dsRNA of 24-30 nucleotides that interacts with a target RNA sequence to direct the cleavage of the target RNA.
  • Lipid particles and more specifically lipid nanoparticles (lipidoid nanoparticles, LNPs), as a class, have been demonstrated as being effective in delivering nucleic acids and nucleic acid analogs to cells, often in vivo (see, e.g., Kulkarni, JA, et al.,“Lipid Nanoparticles Enabling Gene Therapies: From Concepts to Clinical Utility” Nucleic Acid Ther. 2018 Jun;28(3): 146-157. doi:
  • lipid particle e.g.
  • a lipid nanoparticle comprises a mixture of an ionizable and/or cationic lipid, cholesterol or a cholesterol analogue, a helper lipid, such as DSPC, DOPC (dioleoyl phosphatidylcholine), DSPE (distearoyl phosphatidylethanolamine), or DOPE, a polyethylene glycol)-lipid conjugate (PEG-lipid) or PEG-cholesterol conjugate (PEG-cholesterol), and a polyanionic therapeutic agent, such as a nucleic acid or nucleic acid analog.
  • a helper lipid such as DSPC, DOPC (dioleoyl phosphatidylcholine), DSPE (distearoyl phosphatidylethanolamine), or DOPE
  • PEG-lipid polyethylene glycol
  • PEG-cholesterol PEG-cholesterol
  • a polyanionic therapeutic agent such as a nucleic acid or nucleic acid analog.
  • the PEG-lipid or PEG-cholesterol may be modified with a targeting moiety, such as N-acetylgalactosamine (GalNAc) for liver targeting, or another ligand or binding reagent, such as an antibody or antibody fragment.
  • a targeting moiety such as N-acetylgalactosamine (GalNAc) for liver targeting, or another ligand or binding reagent, such as an antibody or antibody fragment.
  • the lipid particle or lipid nanoparticle includes, along with the nucleic acid, e.g., siRNA, heptatriaconta-6,9,28,31 -tetraen-19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA, MC3, see, U.S. Patent Application Publication No.
  • 2013/0245107 A1 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and polyethylene glycol-dimyristolglycerol (PEG-DMG), e.g., at a molar ratio of 50:10:38.5:1 .5 (see, e.g., Tam, Y.Y.C. et at. “Advances in Lipid Nanoparticles for siRNA Delivery” Pharmaceutics 2013, 5, 498-507; doi:10.3390/pharmaceutics5030498).
  • PEG-DMG polyethylene glycol-dimyristolglycerol
  • the amino group of MC3 is protonated and interacts with the negatively charged nucleic acids, thereby promoting the self-assembly of the formulation components into particles, such as nanoparticles, encapsulating the siRNA.
  • a polyanionic helper polymer such as a negatively-charged polymer
  • significantly lower amounts of siRNA is required to achieve the same therapeutic effect as the same lipid nanoparticles that do not comprise the polyanionic helper polymer.
  • Ionizable cationic lipid Ionizable cationic lipid
  • Useful ionizable or cationic lipids include a lipid moiety attached to (covalently-linked to) an ionizable or cationic group.
  • Effective ionizable lipids tend to be positively charged at low pH (which aids in RNA complexation when it is carried out in acidic buffer) but are neutral at physiological pH (for reduced toxicity post injection).
  • cellular uptake via endocytosis deposits nanoparticles into endosomal compartments, which slowly reduce their pH from approximately 6.8 to 4.5 as they morph into lysosomes.
  • 9,439,968 discloses tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl)bis(propane-3, 1 - diyl))bis(azanetriyl)) tetrapropionate (3060iio), which is described in the examples below.
  • the ionizable or cationic lipid is DLin-MC3-DMA, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3]-dioxolane (DLin-KC2-DMA, KC2, see, e.g., U.S. Patent No. 8,754,062), or cKK-E12
  • the weight ratio of ionizable lipid to RNA in a typical LNP is between 5:1 and 15:1 .
  • the helper lipid is a cationic, anionic, neutral, or zwitterionic amphiphilic lipid that, along with cholesterol or a cholesterol analog, aids in the molecular packing and stability of the particle.
  • Helper lipids also enhance lipid nanoparticle efficacy by promoting fusion with both cell and endosomal membranes, facilitating cell uptake and endosomal release.
  • Useful helper lipids include, for example and without limitation, DSPC, DSPE, DOPC, and DOPE. Additional useful helper lipids are known in the art, e.g. phosphatidylcholine lipids.
  • the molar ratio of ionizable lipid to helper lipid in a typical LNP ranges from 1 : 0.001 to 1 : 0.5. In examples, the molar ratio of ionizable lipid to cholesterol or cholesterol analogue in a typical LNP ranges from 1 : 0.75 to 1 : 1.35.
  • Non-limiting examples of cholesterol analogues include: oxidized cholesterol, desmosterol, 7-dehydrocholesterol, ergosterol, lanosterol, ketosterone, cholesterol sulfate, dehydroergosterol, cholestratrienol, 5-cholestene, or pregnenolone.
  • PEG-lipids comprise a polyethylene glycol) moiety attached to one or more lipid moieties, for example and without limitation, with a ceramide, succinoyl, or carbamate linkage.
  • PEG-cholesterol PEGylated cholesterol
  • PEG-lipids and/or PEG-cholesterols form a protective, non-aggregating, non- immunogenic shell around the surface of the LNP.
  • the lipid group may be varied, e.g., in length, to dictate how long the PEG-lipid is associated with the LNP, with longer lipid chains tending to remain associated with the LNP for longer time periods, with shorter lipid chains being useful in providing“diffusible” PEG lipids that diffuse from the lipid nanoparticle quickly to produce an LNP with increased transfection rates in many situations, and when desirable.
  • the PEG group or moiety of the PEG-lipid or PEG-cholesterol typically has a molecular weight ranging from 300 g/mol to 5000 g/mol, e.g., ⁇ 2000 g/mol (referred to as PEG 2000).
  • Non-limiting examples of suitable PEG-lipids include: 1 ,2- Dimyristoyl-sn-Glycero-3-Phosphoethanolamine-N [Methoxy(Polyethylene glycol)- 2000], N-octanoyl-sphingosine-1 - ⁇ succinyl[methoxy(polyethylene glycol)2000] ⁇ , N- palmitoyl-sphingosine-1 - ⁇ succinyl[methoxy(polyethylene glycol)5000] ⁇ , 1 ,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 3000] (ammonium salt), 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-1000] (ammonium salt), and PEG-cholesterol, such as cholesterol-(polyethylene glycol-600).
  • targeting of the LNP may be achieved by including in the LNP a ligand specific to the cells to be targeted attached to a lipid moiety, such as attaching the ligand to a PEG moiety of the PEG-lipid, or attaching the ligand to a lipid moiety via any suitable linker or spacer, as are broadly-known.
  • Suitable ligands include cell penetrating peptides (CPPs), transferrin, folate, oligosaccharides, polysaccharides, and antibodies or other binding reagents, e.g., GalNAc for liver targeting.
  • the term“ligand” refers to a binding moiety for a specific target, referred to as its binding partner.
  • the molecule can be a cognate receptor, a protein a small molecule, a hapten, or any other relevant molecule.
  • the term“antibody” refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. As such, the antibody operates as a ligand for its cognate antigen, which can be virtually any molecule. Natural antibodies comprise two heavy chains and two light chains and are bi-valent.
  • variable regions of heavy and light chain forms a binding site capable of specifically binding an antigen (e.g., a paratope).
  • antigen e.g., a paratope.
  • VH refers to a heavy chain variable region of an antibody.
  • VL refers to a light chain variable region of an antibody.
  • Antibodies may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE.
  • Ligands also referred to as binding reagents, having limited crossreactivity are generally preferred.
  • suitable ligands include, for example, polypeptides, such as for example, antibodies, monoclonal antibodies, or derivatives or analogs thereof, including without limitation: Fv fragments, single chain Fv (scFv) fragments, Fabi fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((ScFv)2 fragments), diabodies, triabodies, tetrabodies, which typically are covalently linked or otherwise stabilized (i.
  • scFv fragments e., leucine zipper or helix stabilized
  • scFv fragments di-scFv (dimeric single-chain variable fragment), single-domain antibody (sdAb), or antibody binding domain fragments and other binding reagents including, for example, bi-specific T-cell engagers (BiTEs), aptamers, template imprinted materials, and organic or inorganic binding elements.
  • BiTEs bi-specific T-cell engagers
  • aptamers e.g., a ligand specifically interacts with a single epitope.
  • a ligand may interact with several structurally related epitopes.
  • the term“antibody fragment” refers to any derivative of an antibody which is less than full-length.
  • the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability.
  • binding reagents including antibody fragments, but are not limited to, Fab, Fab', F(ab')2, Fv, Fd, dsFv, scFv, diabody, triabody, tetrabody, di-scFv (dimeric single-chain variable fragment), bi-specific T-cell engager (BiTE), single-domain antibody (sdAb), or antibody binding domain fragments.
  • the antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody, or it may be recombinantly or partially synthetically produced.
  • the antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multimolecular complex.
  • a functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.
  • Antibody fragments also include miniaturized antibodies or other engineered binding reagents that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigenbinding or epitope-binding sequences and at a minimum any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition (see, e.g., Nelson, AL, “Antibody Fragments Flope and Flype” (2010) MAbs 2(1 ):77-83).
  • the helper polymer is a polyanion, having an overall negative charge, e.g. a pKa of less than 4, e.g., less than 3, or 2, or approximately 1 .
  • a polyanionic polymer may have a hydrocarbon backbone, as with polyvinyl or olefinic polymers and may have the structure -[C-C(R)]-, wherein R is a moiety comprising an acidic group or its conjugate base, such as sulfonate, carboxyl, and phosphinic acid (HOP(O)H-) groups, linked to the backbone either directly or through a C1-C6 aliphatic or aromatic hydrocarbon linker.
  • the hydrocarbon backbone is styrene, where R includes a phenyl moiety, substituted with an acidic group or its conjugate base, such as phosphate, sulfonate, carboxylate, sulphates, alkoxydicyanoethanolates, boronates, phenolates, sulfonamides, sulfonimides, and phosphinic acid.
  • R includes a phenyl moiety, substituted with an acidic group or its conjugate base, such as phosphate, sulfonate, carboxylate, sulphates, alkoxydicyanoethanolates, boronates, phenolates, sulfonamides, sulfonimides, and phosphinic acid.
  • Additional examples of polyanionic polymers useful in the present disclosure include: carboxylate and sulphonate styrene- divinylbenzene copolymer, polyglutamate, and carboxymethylcellulose.
  • the helper polymer has a number average molecular weight (Mri) ranging from 1 kDa to 200 kDa, for example, from 1 kDa to 100 kDa, or from 5 kDa to 75 kDa.
  • the helper polymer is a polystyrene, such as a polystyrene sulfonate having an Mn ranging from 5 kDa to 75 kDa, and a PDI of less than 5.
  • the weight ratio of helper polymer to RNA in a typical LNP is between 1 1 :1 and 0.04 : 1 .
  • the therapeutic agent is an siRNA reagent
  • the helper polymer is an mRNA or other low-PDI (e.g., having a PDI of less than 2) nucleic acid.
  • the mRNA encodes a protein.
  • the protein is a therapeutic protein.
  • the LNP described herein comprises an ionizable or cationic lipid (ICL), cholesterol, a helper lipid, a polyethylene glycol)-lipid conjugate (PEG-lipid), a polyanionic therapeutic agent, and a helper polymer.
  • ICL ionizable or cationic lipid
  • helper lipid lipid
  • PEG-lipid polyethylene glycol-lipid conjugate
  • polyanionic therapeutic agent lipid
  • helper polymer a polyanionic therapeutic agent
  • helper polymer ionizable or cationic lipid
  • the relative ratio of each component will vary depending on the selected constituent. Examples of specific LNP formulations are provided in the examples below.
  • RNA-reagents For RNA-reagents, a suitable weight ratio of total lipid (referring to the sum of the weights of ionizable or cationic lipid (ICL), cholesterol, helper lipid, PEG-lipid, and any other lipid or lipidoid constituents) to RNA, e.g., mRNA or siRNA may be between 10:1 and 30:1.
  • the range of ratios of various constituents of the LNPs for delivery of mRNA and/or siRNA, described herein may be as indicated in Table 1 , providing two ranges (A and B) for each example.
  • Coformulation also improved mRNA delivery, as a 0.5 mg/kg dose of mRNA coformulated with siRNA induced three times the luciferase protein expression compared to when siRNA was not included.
  • RNA drugs we sought to extend the synergy of co-formulated LNPs to formulations encapsulating only a single type of RNA. We accomplished this by substituting the “helper” RNA with a negatively charged polymer, polystyrene sulfonate (PSS). LNPs containing PSS mediated the same level of protein silencing or expression as standard LNPs using 2-3 fold less RNA.
  • PSS polystyrene sulfonate
  • LNPs formulated with and without PSS induced 50% Factor VII silencing at siRNA doses of 0.01 and 0.03 mg/kg, respectively.
  • these studies demonstrate potent co-delivery of siRNA and mRNA and show that inclusion of a negatively charged“helper polymer” enhances the efficacy of LNP delivery systems.
  • RNA drugs including short interfering RNA (siRNA) and messenger RNA (mRNA), can theoretically treat any disease caused by gene dysregulation. Conditions associated with protein overexpression may benefit from siRNA drugs, which inhibit protein production by cleaving mRNA. On the other hand, diseases caused by insufficient protein production are candidates for mRNA therapy. Both types of RNA therapy have made significant translational progress over the past several years, often being delivered in ionizable polymer or lipid nanoparticles. The clinical translation of siRNA therapy hit a major milestone in 2017 with the completion of the first successful Phase 3 clinical trial by Alnylam Pharmaceuticals.
  • RNAs Delivery of both RNAs would enable simultaneous knockdown of undesirable protein(s) and expression of desirable protein(s). Such an approach would apply to many diseases, including liver cancer, which is characterized by the upregulation of oncogenes and downregulation of tumor suppressor genes. Encapsulation of siRNA and mRNA in a single particle guarantees that transfected cells receive both drugs, maximizing the intended therapeutic effect. A single formulation would also reduce production costs and regulatory hurdles compared to a therapy comprising two separate siRNA and mRNA formulations. Given the considerably different molecular characteristics of siRNA and mRNA, successful coformulation requires nanoparticle chemistry that accommodates both therapeutic molecules. For example, siRNA and mRNA have drastically different molecular weights (10 4 vs. 10 6 g/mol), stability, and molecular conformation.
  • Ionizable lipid nanoparticles have shown significant translational promise in the delivery of siRNA and, separately, mRNA.
  • the LNPs used to encapsulate siRNA and mRNA consist of the same four primary components: an ionizable lipid or lipidoid compound, cholesterol, a helper lipid, and polyethylene glycol (PEG)-lipid.
  • PEG polyethylene glycol
  • the ratio of these components, together with the ratio of the ionizable lipid to RNA can significantly alter delivery efficacy in vitro and in vivo.
  • Previous work has demonstrated that siRNA and mRNA are most effectively delivered in distinct LNP formulations.
  • Formulation 5 features a higher lipidoid to RNA ratio and a lower molar percentage of lipidoid in relation to helper lipid, cholesterol, and PEG-lipid. Additionally, the helper lipid shifts from DSPC in Formulation 1 to DOPE in Formulation 5. DSPC contains two saturated aliphatic tails, while DOPE contains a c/s-double bond in each of its two aliphatic tails. This structural difference changes the packing within the lipid nanoparticle and can affect endosomal escape, which is important given the significant molecular differences between siRNA and mRNA.
  • FIG. 1 (A) shows resultant luciferase gene silencing. As expected, control particles containing only mRNA induced no gene knockdown. For particles loaded with siRNA, gene silencing increased with increasing formulation number.
  • mice were injected by tail vein with LNPs loaded with RNA doses of 0.03 mg/kg siFVII and/or 0.5 mg/kg mLuc.
  • RNA doses 0.03 mg/kg siFVII and/or 0.5 mg/kg mLuc.
  • mRNA delivery- mediated luciferase expression was assessed using whole body luminescence imaging.
  • Efficacy of siRNA delivery was measured 48 hours after injection by quantifying serum Factor VII protein levels.
  • Formulations 3-5 loaded with both RNAs produced the highest levels of Factor VII silencing ( ⁇ 90%, circles). This degree of knockdown was markedly better than when siRNA was delivered in Formulation 1 ( ⁇ 50% silencing, triangle) and the mRNA control (0% silencing, square).
  • co-formulated LNPs improved Factor VII silencing from 50 to 90% (open circles) and tripled luciferase expression (squares and Figure 3 (D)).
  • the two treated animal groups received the same total amount of each LNP ingredient, including the lipidoid.
  • RNA concentration used for formulation does not necessarily go hand in hand with in vivo dose.
  • Figure 4 (A) a set of five LNPs were formulated in which the siRNA concentration was held constant at 0.006 mg/mL while the concentration of mRNA was gradually increased from 0 to 0.2 mg/mL. All LNPs were dosed in mice at 0.03 mg/kg of siRNA. The addition of mRNA improved FVII silencing, but only up to a total RNA concentration of 0.106 mg/mL. Beyond that, the benefit of co-formulation diminished and finally disappeared at a total RNA concentration of 0.206 mg/mL.
  • N:P nitrogen to phosphate
  • LNPs co-formulated included higher amounts of lipidoid and other lipid content, as we believed this would be necessary to provide enough material to encapsulate the higher RNA amount. Because an increase in lipidoid concentration can sometimes confer LNP efficacy, we asked if this was the reason for enhanced efficacy of both RNA species upon co-formulation.
  • LNPs formulated with high lipidoid content and without “helper” mRNA only modestly improved FVII protein silencing from 37 to 49% compared to LNPs formulated at a low lipidoid concentration.
  • a marked increase in protein silencing to 87% was observed only when helper mRNA and increased lipidoid were included in the formulation.
  • RNA is a negatively charged biopolymer, it may be possible to replace it with a negatively charged synthetic polymer.
  • uridine homopolymer Poly(U)
  • PSS Poly(sodium 4-sytrenesulfonate)
  • PAA Poly(acrylic acid)
  • Poly(U) which is a polymer of the RNA base uracil, is available at a much lower cost than sequence-specific mRNA given the increased simplicity of synthesis.
  • PSS is a synthetic, biocompatible polymer that is used clinically to treat hyperkalemia and lithium poisoning in humans.
  • PAA another synthetic polymer, has also been used in biomedical applications.
  • PSS best mimics the effect of helper RNA is that it is negatively charged at all pH values relevant to LNP formulation and delivery.
  • Both the PSS monomer and the phosphodiester group in the RNA backbone have pK a values of one.
  • LNPs are formulated under acidic conditions so that the lipidoid amines protonate and subsequently attract the negatively charged RNA. It is possible that the additional negative charge provided by the polyanion promotes the formation of a more stable and/or compact nanoparticle by increasing the electrostatic attraction inside the particle.
  • PAA with a higher pK a of four, may not mediate the same electrostatic effect.
  • Poly(U) while imparting some benefit to siRNA-loaded LNPs, may not facilitate the same intraparticle molecular interactions as mRNA because of its polydispersity (Mw 100 - 1 ,000 kDa).
  • LNPs formulated with PSS and dosed at 0.01 mg/kg achieved the same level of silencing as those formulated without PSS and dosed at 0.03 mg/kg.
  • the incorporation of PSS into an LNP formulation facilitated a three-fold reduction in the amount of siRNA required.
  • the total amount of lipidoid dosed to mice was the same for each group in Figure 9 (C).
  • the efficacy“savings” afforded by PSS will not be offset by increased toxicity due to increased lipid content.
  • PSS did not improve RNA entrapment, and nanoparticle size did not correlate with efficacy (Figure 12 (A-B)). None of the treatments resulted in significant weight loss compared to the untreated mice ( Figure 12 (C)).
  • Cholesterol was purchased from Sigma Aldrich (St. Louis, MO), and distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), and 1 ,2-Dimyristoyl-sn-Glycero-3- Phosphoethanolamine-N [Methoxy(Polyethylene glycol)- 2000] (14:0 PEG2000-PE) were obtained from Avanti Polar Lipids (Alabaster, AL). HeLa cells were purchased from American Type Culture Collection (Manassas, VA).
  • Dulbecco Modified Eagles Media (DMEM), trypsin, penicillin/streptomycin, phosphate buffered saline (PBS), and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific (Waltham, MA).
  • DMEM Modified Eagles Media
  • PBS phosphate buffered saline
  • FBS fetal bovine serum
  • siRNA for Luciferase was purchased from Dharmacon (Lafayette, CO).
  • Anti-Factor VII siRNA was custom ordered from Sigma Aldrich with a sense sequence of 5’-GGAucAucucAAGucuuAcT*T-3’ and an antisense sequence of 5’-GuAAGAcuuGAGAuGAuccT*T-3’, where lower case nucleotides are 2’-fluoro- modified and asterisks indicate phosphorothioate linkages.
  • Clean CapTM mCHERRY and firefly luciferase mRNA were purchased from TriLink Biotechnologies (San Diego, CA).
  • Poly(S Mw 70,000 and 6,800), poly(acrylic acid) (PAA Mv -450,000), and polyuridylic acid potassium salt (Poly(U) Mw 100 - 1 ,000 kDa) were purchased from Sigma Aldrich.
  • LNPs were prepared as previously described.
  • the lipidoid 3060no was synthesized by Michael addition of isodecyl acrylate to 3,3’-diamino-N- methyldipropylamine.
  • the fully-substituted version which was isolated using a Teledyne Isco chromatography system, was used for all experiments.
  • LNPs were formulated by first dissolving the lipidoid, cholesterol, DSPC, DOPE, and PEG2000 in ethanol. A lipid solution was made by mixing these components according to the molar ratios in Table 2 in 90% ethanol and 10% 10 nM sodium citrate (by volume).
  • RNA siRNA and/or mRNA
  • RNA was diluted in 10 nM sodium citrate to achieve a final lipidoid:RNA weight ratio between 5:1 and 10:1 .
  • Rapid pipet mixing was used for spontaneous formation of the LNPs.
  • LNPs were dialyzed for one hour in PBS to remove ethanol.
  • LNPs were diluted in PBS to achieve the desired final concentration.
  • the nanoparticles were diluted to 1 pg/mL total RNA.
  • LNPs were characterized for RNA entrapment using a Quant-iTTM Ribogreen® Assay (Invitrogen, Carlsbad, CA) according to the manufacture’s protocol.
  • Dynamic light scattering (DLS) was used to characterize LNP size and polydispersity (PDI) on a Malvern Zetasizer Nano (Malvern Instruments, UK).
  • HeLa cells stably modified to express firefly and Renilla luciferase were grown in DMEM supplemented with 100 ml/L of FBS, 10 lU/mL of penicillin, and 10 mg/mL of streptomycin. The cells were incubated at 37 °C in a 5% CO2 environment and subcultured by partial digestion with 0.25% trypsin and ethylenediaminetetraacetic acid. Passages 10-30 were used for experiments. HeLa cells were seeded at 15,000 cells per well in a black 96 well plate.
  • LNPs were made according to the formulations in Table 2 with either siRNA, mRNA, or siRNA + mRNA at RNA concentrations of 0.0125 mg/mL siRNA and 0.05 mg/mL mRNA.
  • siRNA was specific for firefly luciferase and mRNA encoded mCherry. LNPs were delivered to luciferase-expressing HeLa cells at a dose of 10 nM siRNA and/ or 100 ng ( ⁇ 1 .6 nM) mRNA for 24 hours. Resultant mCHERRY fluorescence (ex:587 nm/ em:615 nm) was measured 24 hours after transfection.
  • luciferase activity was quantified using a Dual-Glo ® Luciferase Assay Kit (Promega, Madison, Wl) according to the manufacture’s protocol. After the first Luciferase Assay Kit reagent was added, the lysed cells were transferred to a white plate to measure luminescence. Renilla luciferase activity served as a control.
  • LNPs for in vivo experiments were formulated with either anti-Factor VII siRNA (siFVII), mRNA encoding luciferase (mLuc), or a combination of the two and administered to mice via tail-vein injection.
  • siFVII anti-Factor VII siRNA
  • mLuc mRNA encoding luciferase
  • luciferase activity was assessed by administering an intraperitoneal injection of D-Luciferin substrate (130 pL at 30 mg/mL in PBS). 15 minutes later, luminescence was measured by whole mouse imaging using an IVIS (Perkin Elmer, MA) and quantified using Living Image software (Perkin Elmer).
  • Factor VII expression was measured 48 hours after LNP injection from a submandibular blood sample.
  • a BIOPHEN FVII assay was used according to the manufacture’s protocol (Aniara, OH). Mice were also weighed 48 hours post-injection, as significant weight loss may indicate LNP toxicity.
  • LNPs were formulated according to the specifications in Table 2 at concentrations of 0.006 mg/ml siFVII and/or 0.1 mg/ml mLuc. Mice were dosed with 0.03 mg/kg siRNA and/or 0.5 mg/kg mRNA.
  • Figures 2 (D) 4, and 9, all LNPs were made using Formulation 4.
  • the siRNA formulation concentration was kept constant at 0.006 mg/mL while mRNA concentration varied from 0 - 0.2 mg/mL. This corresponded to a final dose in mice of 0.03 mg/kg siRNA and 0 - 1 mg/kg mRNA.
  • the LNP mRNA formulation concentration was kept constant at 0.1 mg/mL and the siRNA concentration varied from 0 - 0.08 mg/mL. Mice were dosed with 0.5 mg/kg mRNA and 0 - 0.4 mg/kg siRNA.
  • lipid and RNA solutions were prepared in ethanol and 10 nM sodium citrate, respectively, as described in the Lipid Nanoparticle Formulation section above.
  • Negatively charged polymer Poly(U), PSS, or PAA
  • PBS Negatively charged polymer
  • PAA PAA
  • LNPs containing siRNA as the active drug were formulated at 0.006 mg/mL siFVII and with either mLuc (0.1 mg/mL), PSS (Mw 70 kDa, 0.064 mg/mL), Poly(U) (Mw 100 - 1 ,000 kDa, 0.1 mg/mL), or PAA (Mv 450 kDa, 0.1 mg/mL) while keeping the lipidoid concentration constant at 0.93 mg/mL. All mice were dosed with LNP formulations containing 0.03 mg/kg siFVII.
  • LNPs containing mRNA as the active drug were formulated at 0.1 mg/mL mLuc and with either siFVII (0.006 mg/mL), PSS (Mw 6.8 kDa, 0.004 mg/mL), Poly(U) (Mw 100 - 1 ,000 kDa, 0.005 mg/mL), or PAA (Mv 450 kDa, 0.0013 mg/mL) while keeping the lipidoid concentration constant at 0.93 mg/mL. All mice were dosed with LNP formulations containing 0.5 mg/kg mLuc.
  • the negative polymer concentrations were decided based on maintaining the same amount of total negative charge as when the LNPs are made with 0.006 mg/mL siRNA and 0.1 mg/mL mRNA.
  • siFVII was used as the active drug and formulated at either 0.004 mg/ml_ siFVII and PSS (70 kDa, 0.066 mg/ml_), 0.002 mg/ml_ and PSS (70 kDa, 0.067 mg/ml_) or 0.001 mg/ml_ siFVII and PSS (70 kDa, 0.068 mg/ml_) and IV-injected to mice at doses of 0.005 mg/kg - 0.3 mg/kg.
  • the negative polymer concentrations for these experiments were selected to maintain the same total negative charge as when the LNPs were formulated with 0.006 mg/ml_ siRNA and 0.1 mg/ml_ mRNA.

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Abstract

L'invention concerne des particules lipidiques améliorées, des méthodes de fabrication de ces particules lipidiques, ainsi que des méthodes d'utilisation de celles-ci, notamment pour l'apport de réactifs thérapeutiques ARN, tels que ARNsi et ARNm.
PCT/US2019/027885 2018-04-17 2019-04-17 Apport de médicament sous forme de nanoparticules lipidiques améliorées, à l'aide d'un polymère chargé négativement WO2019204451A1 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
CN113908292A (zh) * 2021-10-13 2022-01-11 南京吉迈生物技术有限公司 靶向物介导的核酸纳米制剂及其制备方法
WO2022236093A1 (fr) * 2021-05-07 2022-11-10 Carnegie Mellon University Administration d'arnm médiée par des nanoparticules lipidiques au pancréas
WO2022260772A1 (fr) * 2021-06-09 2022-12-15 Carnegie Mellon University Formulations de nanoparticules lipidiques pour administration gastro-intestinale
WO2023201295A1 (fr) * 2022-04-14 2023-10-19 The Trustees Of The University Of Pennsylvania Lipidoïdes biodégradables et compositions et leurs procédés utilisation pour une administration ciblée au foie

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US8956572B2 (en) * 2011-11-04 2015-02-17 Nitto Denko Corporation Single use system for sterilely producing lipid-nucleic acid particles
US9035039B2 (en) * 2011-12-22 2015-05-19 Protiva Biotherapeutics, Inc. Compositions and methods for silencing SMAD4
US20170021036A1 (en) * 2013-06-22 2017-01-26 Ethris Gmbh Compositions for introducing rna into cells
US20170210698A1 (en) * 2015-09-17 2017-07-27 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents

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Publication number Priority date Publication date Assignee Title
US8956572B2 (en) * 2011-11-04 2015-02-17 Nitto Denko Corporation Single use system for sterilely producing lipid-nucleic acid particles
US9035039B2 (en) * 2011-12-22 2015-05-19 Protiva Biotherapeutics, Inc. Compositions and methods for silencing SMAD4
US20170021036A1 (en) * 2013-06-22 2017-01-26 Ethris Gmbh Compositions for introducing rna into cells
US20170210698A1 (en) * 2015-09-17 2017-07-27 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022236093A1 (fr) * 2021-05-07 2022-11-10 Carnegie Mellon University Administration d'arnm médiée par des nanoparticules lipidiques au pancréas
WO2022260772A1 (fr) * 2021-06-09 2022-12-15 Carnegie Mellon University Formulations de nanoparticules lipidiques pour administration gastro-intestinale
CN113908292A (zh) * 2021-10-13 2022-01-11 南京吉迈生物技术有限公司 靶向物介导的核酸纳米制剂及其制备方法
WO2023201295A1 (fr) * 2022-04-14 2023-10-19 The Trustees Of The University Of Pennsylvania Lipidoïdes biodégradables et compositions et leurs procédés utilisation pour une administration ciblée au foie

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