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WO2024236361A1 - Compositions et procédés d'administration d'acides nucléiques à des cellules - Google Patents

Compositions et procédés d'administration d'acides nucléiques à des cellules Download PDF

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Publication number
WO2024236361A1
WO2024236361A1 PCT/IB2024/000233 IB2024000233W WO2024236361A1 WO 2024236361 A1 WO2024236361 A1 WO 2024236361A1 IB 2024000233 W IB2024000233 W IB 2024000233W WO 2024236361 A1 WO2024236361 A1 WO 2024236361A1
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Prior art keywords
lipid
lnp
composition
sterol
helper
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English (en)
Inventor
Mohammad Parvez ALAM
Roshan Padmashali Manohara PADMASHALI
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Takeda Pharmaceutical Co Ltd
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Takeda Pharmaceutical Co Ltd
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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

Definitions

  • nucleic acids The effective targeted delivery of biologically active substances such as nucleic acids by lipid nanoparticle represents a continuing medical challenge.
  • the delivery of nucleic acids to cells is made difficult by potential toxicity and instability.
  • the present invention provides a lipid nanoparticle (LNP) composition
  • a cationic ionizable lipid comprising: a. a cationic ionizable lipid; b. a helper lipid, wherein the helper lipid is selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), and 1,2- dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC);
  • DSPC l,2-distearoyl-sn-glycero-3-phosphocholine
  • DOPE 1,2-dioleoyl-sn-g
  • a sterol wherein the sterol is selected from the group consisting of cholesterol, P-sitosterol, campesterol, ergosterol, phytosterols (e.g., stigmastanol), and fucosterol;
  • a conjugated-lipid wherein the conjugated-lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG)-1000K, PEG-DMG-2000K, PEG-DMG-3000K, PEG-DMG-4000K and PEG-DMG-5000K; and e.
  • nucleic acid wherein the nucleic acid is DNA and at least about 2 kilobases (kb), at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, or at least about 10 kb.
  • kb 2 kilobases
  • the cationic ionizable lipid is (6Z,9Z,28Z,31Z)-Heptatriaconta- 6,9,28,3 l-tetraen-19-yl 4-(Dimethylamino)butanoate (DLin-MC3-DMA), or 2-(2,2- Di((9Z, 12Z)-octadeca-9, 12-dien-l -yl)- 1,3 -di oxolan-4-yl)-N,N-dimethyl ethanamine (DLin- KC2-DMA).
  • DLin-MC3-DMA 2-(2,2- Di((9Z, 12Z)-octadeca-9, 12-dien-l -yl)- 1,3 -di oxolan-4-yl)-N,N-dimethyl ethanamine
  • the cationic ionizable lipid is DLin-MC3-DMA.
  • the cationic ionizable lipid is DLin-K2C-DMA.
  • the helper lipid is DOPC.
  • the sterol is cholesterol
  • the molar ratio of the cationic ionizable lipid, the helper lipid, the sterol, and the conjugated lipid is about 20%-80% : 5%-20% : 36%-39% : l%-4% (mol) respectively of the total lipid present in the LNP composition.
  • the molar ratio of the cationic ionizable lipid, the helper lipid, the sterol, and the conjugated lipid is about 50% : 10% : 38.5% : 1.5% (mol) respectively of the total lipid present in the LNP composition.
  • the cationic ionizable lipid is DLin-MC3-DMA
  • the helper lipid is DOPC
  • the sterol is cholesterol
  • the conjugated lipid is PEG-DMG
  • the molar ratio of the cationic ionizable lipid, the helper lipid, the sterol, and the conjugated lipid is about 50% : 10% : 38.5% : 1.5% (mol) respectively of the total lipid present in the LNP composition.
  • the cationic ionizable lipid is DLin-KC2-DMA
  • the helper lipid is DOPC
  • the sterol is cholesterol
  • the conjugated lipid is PEG-DMG
  • the molar ratio of the cationic ionizable lipid, the helper lipid, the sterol, and the conjugated lipid is about 50% : 10% : 38.5% : 1.5% (mol) respectively of the total lipid present in the LNP composition.
  • the LNP has a negative zeta potential.
  • the LNP has a zeta potential between about -0.1 mV to about -15 mV, about -1.0 mV to about -10 mV, about -2.0 mV to about -8.0 mV, about -4.0 mV to about -6.0 mV, or about -5.0 mV to about -10 mV.
  • the LNP has a zeta potential of at least -0.1 mV, at least -0.5 mV, at least -1.0 mV, at least -2.0 mV, at least -5.0 mV, at least -10 mV, or at least -15 mV.
  • the LNP has a zeta potential of at most -0.1 mV, at most -0.5 mV, at most -1.0 mV, at most -2.0 mV, at most -5.0 mV, at most -10 mV, or at most -15 mV.
  • the LNP composition has a polydispersity index (PDI) of less than 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1.
  • PDI polydispersity index
  • the LNP composition has a nitrogen/phosphate (N/P) ratio is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or about 15. In one embodiment, the N/P ratio is about 4.
  • the present invention provides a pharmaceutical composition, comprising the LNP composition as disclosed herein, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises a cryoprotectant.
  • the cryoprotectant is a sugar, starch, polyvinyl pyrrolidone, polyethylene oxide, or a combination thereof.
  • the pharmaceutical composition comprises a sugar.
  • the sugar is a disaccharide.
  • the sugar is selected from the group consisting of lactose, sucrose, trehalose, and a combination thereof.
  • amount of sugar is between about 0% w/v and about 40% w/v. In one embodiment, the amount of sugar is at least 5% w/v, at least 10% w/v, at least 20% w/v, or at least 30%. In one embodiment, the amount of sugar is about 20% w/v.
  • the present invention provides a method of producing a pharmaceutical composition, comprising a) producing a lipid nanoparticle (LNP) by mixing a lipid mixture and an aqueous solution, wherein the lipid mixture comprises: i. a cationic ionizable lipid; ii.
  • LNP lipid nanoparticle
  • helper lipid wherein the helper lipid is selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), and l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC); iii.
  • DSPC l,2-distearoyl-sn-glycero-3-phosphocholine
  • DOPE dioleoyl-sn-glycero-3- phosphoethanolamine
  • DOPC 1,2- dimyristoyl-sn-glycero-phosphocholine
  • DPPC 1,2- dimyristoyl-sn-gly
  • a sterol wherein the sterol is selected from the group consisting of cholesterol, P-sitosterol, campesterol, ergosterol, phytosterols (e.g., stigmastanol), and fucosterol;
  • a conjugated lipid wherein the conjugated lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG)-1000K, PEG-DMG-2000K, PEG-DMG-3000K, PEG-DMG-4000K and PEG-DMG-5000K; and the aqueous solution comprises: iv.
  • nucleic acid wherein the nucleic acid is DNA and at least about 2 kilobases (kb), at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, or at least about 10 kb; and b) mixing the LNPs produced in step a) and a pharmaceutically acceptable carrier.
  • kb kilobases
  • the cationic ionizable lipid is (6Z,9Z,28Z,31Z)-Heptatriaconta- 6,9,28,3 l-tetraen-19-yl 4-(Dimethylamino)butanoate (DLin-MC3-DMA), or 2-(2,2- Di((9Z, 12Z)-octadeca-9, 12-dien-l -yl)- 1,3 -di oxolan-4-yl)-N,N-dimethyl ethanamine (DLin- KC2-DMA).
  • DLin-MC3-DMA 2-(2,2- Di((9Z, 12Z)-octadeca-9, 12-dien-l -yl)- 1,3 -di oxolan-4-yl)-N,N-dimethyl ethanamine
  • the cationic ionizable lipid is DLin-MC3-DMA.
  • the cationic ionizable lipid is DLin-KC2-DMA.
  • the helper lipid is DOPC.
  • the sterol is cholesterol
  • the pharmaceutical composition comprises a sugar.
  • the molar ratio of the cationic ionizable lipid, the helper lipid, the sterol, and the conjugated lipid is about 20%-80% : 5%-20% : 36%-39% : l%-4% (mol) of the total lipid present in the LNP composition.
  • the molar ratio of the cationic ionizable lipid, the helper lipid, the sterol, and the conjugated lipid is about 50% : 10% : 38.5% : 1.5% (mol) of the total lipid present in the LNP composition.
  • the molar ratio of the cationic ionizable lipid, the helper lipid, the sterol, and the conjugated lipid is about 50% : 10% : 38.5% : 1.5% (mol) of the total lipid present in the LNP composition.
  • the cationic ionizable lipid is DLin-MC3-DMA
  • the helper lipid is DOPC
  • the sterol is cholesterol
  • the conjugated lipid is PEG-DMG-2000K
  • the molar ratio of the cationic ionizable lipid, the helper lipid, the sterol, and the conjugated lipid is about 50% : 10% : 38.5% : 1.5% (mol) of the total lipid present in the LNP composition.
  • the cationic ionizable lipid is DLin-KC2-DMA
  • the helper lipid is DOPC
  • the sterol is cholesterol
  • the conjugated lipid is PEG-DMG-2000K
  • the molar ratio of the cationic ionizable lipid, the helper lipid, the sterol, and the conjugated lipid is about 50% : 10% : 38.5% : 1.5% (mol) of the total lipid present in the LNP composition.
  • the present invention provides a method of treating a disease or disorder, the method comprising administering the LNP composition as disclosed herein, the pharmaceutical composition as disclosed herein, or the pharmaceutical composition produced by the method as disclosed herein to a subject in need thereof.
  • the disease or disorder is a lysosomal storage disease, an inborn errors of metabolism (IEM) or a hematological disorder.
  • IEM inborn errors of metabolism
  • the LNP composition as disclosed herein, the pharmaceutical composition as disclosed herein, or the pharmaceutical composition produced by the method as disclosed herein is administered intravenously.
  • the LNP composition of as disclosed herein, the pharmaceutical composition as disclosed herein, or the pharmaceutical composition produced by the method as disclosed herein is administered subcutaneously or intramuscularly.
  • the present invention provides a method of optimizing a Lipid nanoparticle (LNP) comprising steps of (1) screening LNPs in vitro and (2) screening LNPs in vivo using immune deficient animal, optionally repeating the step (1) and the step (2).
  • LNP Lipid nanoparticle
  • FIGs. 1A-1C Physico-Chemical Properties of LNP-npDNA(ATP7B) Containing MC3/Lipidl/KC2 Ionizable Lipids in Huh7 Cells.
  • FIG. 1A depicts LNP-npDNA(ATP7B) physio-chemical properties including particle size and polydispersity index (PDI).
  • FIG. IB depicts dependence of particle size (bar) and encapsulation efficiency (star symbol) on N/P ratio and ionizable lipids.
  • FIG. 1C depicts dependence of zeta potential on N/P ratio and ionizable lipids.
  • FIGs. 2A-2B Impact of LNP-npDNA Formulations at Different N/P Ratio on Transgene Expression in Huh7 Cells.
  • FIG. 2A depicts percentage of viable cells as function of N/P ratio following incubation with LNP-npDNA(CAG-Luc) containing MC3 as ionizable-lipid.
  • FIG. 2B depicts luciferase expression as function of N/P ratio following incubation with LNP-npDNA(CAG-Luc) containing MC3 as ionizable-lipid.
  • FIG. 3A-3C Impact of LNP-npDNA Formulations Containing Varying Ionizable Lipids at Different N/P Ratio on Transgene Expression in Huh7 Cells.
  • FIG. 3A depicts percentage of viable cells as function of N/P ratio following incubation with LNP- npDNA(ATP7B) containing ionizable-lipids shown in legend.
  • FIG. 3B depicts quantification of DNA1 expression as function of N/P ratio following incubation with LNP- npDNA(ATP7B) containing ionizable-lipids shown in legend.
  • FIG. 3C depicts DNA1 expression as function of N/P ratio following incubation with LNP-npDNA(ATP7B) containing ionizable-lipids shown in FIG. 3B.
  • Treatment of Huh7 cells were performed using 50 pg/mL stock instead of 25 pg/mL stock for other groups.
  • FIGs. 4A-4C Physico-Chemical Properties and Cell Viability of LNP - npDNA(ATP7B) Systems Containing MC3 and Different Helper Lipids in Huh7 Cells.
  • FIG. 4A depicts LNP-npDNA(ATP7B) physico-chemical properties, including particle size and PDI.
  • FIG. 4B depicts dependence of particle size (bar) and encapsulation efficiency (star symbol) on N/P ratio and helper lipids.
  • FIG. 4C depicts cell viability as function of on N/P ratio and helper lipids.
  • FIG. 5 depicts In-vitro hATP7B protein expression.
  • FIGs. 6A-6B LNP-npDNA(CAG-Luc) System Containing Lipid 1 Ionizable Lipid Exhibit Cell Viability and Transfection Dose Dependence in Huh7 Cells.
  • FIG. 6A depicts cell viability dose response.
  • FIG. 6B depicts impact of LNP-npDNA(C AG- Luc) dose on transgene expression.
  • FIGs. 7A-7C Amino Lipid Comparison with Optimal Helper Lipid/(N/P) Combination.
  • FIG. 7A depicts in vitro cell viability with different LNP formulations and payloads LSPi-hATP7b(wt), LSPi-hATP7b(CodOpt), LSPi-hATP7b(Trunc), or LSP 2 - eGFP(wt).
  • FIG. 7B depicts in vitro protein expression of formulations depicted in FIG. 7A.
  • FIG. 7C depicts in vitro eGFP with LNP formulations including DOPC.
  • FIG. 8. depicts in vivo evaluation of DOPC containing LNP-DNA formulations with ATP7B and eGFP payload nanoplasmids.
  • FIGs. 9A-9F in vivo evaluation of DOPC containing LNP-DNA formulations with ATP7B and eGFP payload nanoplasmids in mice.
  • FIG. 9A depicts protein expression of hATP7B;
  • FIG. 9B depicts the protein levels of hATP7B;
  • FIG. 9C depicts mRNA levels of hATP7B;
  • FIG. 9D depicts npDNA levels.
  • FIG. 9E depicts eGFP protein levels and
  • FIG. 9F depicts eGFP mRNA levels.
  • FIG. 10 depicts in vivo evaluation in immune deficient mice of DOPC containing LNP-DNA formulations with multiple nanoplasmid constructs of varying transgene sizes and different promoter sequences.
  • FIGs. 11A-11B depicts substantial presence of npDNA (FIG. 11A) and mRNA (FIG. 11B) copies from delivered constructs using Lipid 1 -DOPC formulations in mice at Day 7 and 28 post dose.
  • FIGs. 12A-12D Large transgene expression delivered via LNP formulations in mice.
  • FIG. 12A depicts western blot of hATP7B (approx. 1450 aa in size) expression and quantification at day 7.
  • FIG. 12B depicts quantification of FVII-BDD expression in immune deficient (ID) mice.
  • FIG. 12C depicts expression of eGFP in mice.
  • FIG. 12D depicts eGFP expression shown by immunohistochemistry.
  • FIGs. 13A-13D Small transgene expression (expressed proteins approx. 350-450 amino acids in length) delivered via LNP formulations in mice.
  • FIG. 13A depicts expression of PAH under the control of promoters prTTR or prCB shown by immunohistochemistry.
  • FIG. 13B depicts quantification of protein expression of OTC under the control of prTTR.
  • FIG. 13C depicts expression of OTC shown by immunohistochemistry.
  • FIG. 13D depicts quantification of protein expression of GLA over 28 days.
  • FIG. 14 depicts protein expression of ATP7B, PAH, eGFP, OTC, or hFVIII in treated mice shown by immunohistochemistry.
  • FIGs. 15A-15D depict protein expression of ATP7B in liver in wt mice compared to ATP7B expression levels in human liver.
  • FIG. 15B depicts the quantification of protein expression levels shown in FIG. 15A.
  • FIG. 15C depicts expression of GLA in wt mice compared to the saline control.
  • FIG. 15D depicts expression of FVIII-BODD-SQ in wt and immune deficient (ID) mice.
  • FIG. 16 depicts the scheme of optimizing and screening LNP.
  • compositions, methods, and respective component(s) thereof are used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.
  • consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • purify means to make substantially pure or clear from unwanted components, material defilement, admixture or imperfection.
  • the terms “significant” or “significantly” are used synonymously with the term “substantially.”
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • stable refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.
  • stabilize and “stabilized,” means to make or become stable.
  • the present invention provides new compositions of matter, comprising a lipid nanoparticle comprising a. a cationic ionizable lipid, wherein the cationic ionizable lipid is (6Z,9Z,28Z,3 lZ)-Heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(Dimethylamino)butanoate (DLin- MC3-DMA), or 2-(2,2-Di((9Z,12Z)-octadeca-9,12-dien-l-yl)-l,3-dioxolan-4-yl)-N,N- dimethylethanamine (DLin-KC2-DMA); b.
  • a cationic ionizable lipid wherein the cationic ionizable lipid is (6Z,9Z,28Z,3 lZ)-Heptatriaconta-6,9,28,31-tetraen- 19-y
  • helper lipid wherein the helper lipid is selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), and 1,2- dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC); c.
  • DSPC l,2-distearoyl-sn-glycero-3-phosphocholine
  • DOPE 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine
  • DOPC 1,2-dioleoyl-sn-glycero-3- phosphocholine
  • DMPC 1,2-dimyristoyl-sn-g
  • a sterol wherein the sterol is selected from the group consisting of cholesterol, P-sitosterol, campesterol, ergosterol, phytosterols (e.g., stigmastanol), and fucosterol;
  • a conjugated lipid wherein the conjugated lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG)-1000K, PEG-DMG-2000K, PEG-DMG-3000K, PEG-DMG-4000K, and PEG-DMG-5000K; and e.
  • nucleic acid wherein the nucleic acid is DNA and at least 2 kilobases (kb), at least 3 kb, at least 4 kb, at least 5 kb, at least 5 kb, at least 7 kb, at least 8 kb, at least 9 kb, or at least 10 kb.
  • kb kilobases
  • lipid nanoparticle refers to a structure comprising an internal lipophilic core surrounded by a hydrophilic phase encapsulating the core.
  • the ionic interaction resulting from the different lipophilic and hydrophilic components of the nanoparticle generates independent and observable physical characteristics, the mean size of which is equal to or less than 1 pm, i.e., a mean size comprised between 1 and 1000 nm.
  • LNP may comprise a cationic ionizable lipid, a helper lipid, a sterol and conjugated lipid as a lipid.
  • Average size is understood as the average diameter of the population of nanoparticles comprising the lipophilic phase and the hydrophilic phase.
  • the mean size of these systems can be measured by standard methods known by the person skilled in the art, and which are described, for example, in the experimental part below.
  • ionizable lipid or “cationic ionizable lipid” and “amino lipids” are used herein interchangeably and refer to lipids having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH.
  • physiological pH e.g., pH 7.4
  • second pH preferably at or above physiological pH.
  • ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7.
  • the cationic ionizable lipid used in the lipid nanoparticle is not particularly limited.
  • the cationic ionizable lipid can be selected appropriately from the cationic ionizable lipids known by the person skilled in the art.
  • cationic lipids described in WO 2015/011633, WO 2016/021683, WO 2011/153493, WO 2013/126803, WO 2010/054401, WO 2010/042877, WO 2016/104580, WO 2015/005253, WO 2014/007398, WO 2017/117528, WO 2017/075531, WO 2017/00414, WO 2015/199952, US 2015/0239834, WO 2019/131839, or WO 2020/032184 can be in the lipid nanoparticles.
  • the cationic ionizable lipid is (6Z,9Z,28Z,3 lZ)-Heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(Dimethylamino)butanoate (DLin- MC3-DMA), or 2-(2,2-Di((9Z,12Z)-octadeca-9,12-dien-l-yl)-l,3-dioxolan-4-yl)-N,N- dimethylethanamine (DLin-KC2-DMA).
  • the cationic ionizable lipid is DLin-MC3-DMA.
  • the cationic ionizable lipid is DLin-KC2-DMA.
  • helper lipid also known as “neutral lipid”, refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.
  • such lipids include, but are not limited to, phosphotidylcholines such as l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-Dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), l,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1- Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), phophatidyl ethanolamines such as l,2-Dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), sphingomyelins (SM), ceramides, steroids such as sterols and their derivatives.
  • Neutral lipids may be synthetic or naturally derived.
  • structural lipid refers to sterols and also to lipids containing sterol moieties. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle.
  • Structural lipids can include, but are not limited to, cholesterol, P-sitosterol, campesterol, fucosterol, ergosterol, phytosterols (e.g., stigmastanol), fecosterol, ergosterol, bassicasterol, tomatidine, tomatine, ursolic, alphatocopherol, and mixtures thereof.
  • the structural lipid is cholesterol.
  • conjugated lipid refers to a lipid molecule conjugated with a non-lipid molecule, such as a PEG (e.g., PEG-lipid), polyoxazoline, polyamide, or polymer (e g., cationic polymer).
  • the conjugated lipid is a PEG-lipid and is selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG)-1000K, PEG-DMG-2000K, PEG-DMG-3000K, PEG-DMG-4000K, and PEG-DMG-5000K.
  • the conjugated lipid is PEG-DMG-2000K.
  • the cationic ionizable lipid is between about 20% to about 80%, about 25% and about 80%, about 30% and about 80%, about 35% and about 80%, about 40% and about 80%, about 45% and about 80%, about 50% and 80%, about 55% and about 80%, about 60% and about 80%, about 65% and about 80%, about 70% and about 80%, about 75% and about 80%, about 20% to about 75%, about 25% and about 75%, about 30% and about 75%, about 35% and about 75%, about 40% and about 75%, about 45% and about 75%, about 50% and 75%, about 55% and about 75%, about 60% and about 75%, about 65% and about 75%, about 70% and about 75%, about 20% to about 70%, about 25% and about 70%, about 30% and about 70%, about 35% and about 70%, about 40% and about 70%, about 45% and about 70%, about 50% and 70%, about 55% and about 70%, about 60% and about 70%, about 65% and about 70%, about 20% to about 70%, about 25% and about
  • the cationic ionizable lipid is about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80% of the total lipid in the lipid nanoparticle. In some embodiments, the cationic ionizable lipid is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% of the total lipid in the lipid nanoparticle.
  • the helper lipid is between about 5% and about 20%, about 10% and about 20%, about 15% and about 20%, about 5% and about 15%, about 10% and about 15%, or about 5% and about 10% of the total lipid in the lipid nanoparticle. In some embodiments, the helper lipid is about 5%, about 10%, about 15%, or about 20% of the total lipid in the lipid nanoparticle. In some embodiments, the helper lipid is at least 5%, at least 10%, at least 15%, or at least 20% of the total lipid in the lipid nanoparticle.
  • the sterol is between about 36% and about 39%, about 36% and 38.5%, about 36% and about 38%, about 36% and about 37.5%, about 36% and about 37%, about 36% and about 36.5%, about 36.5% and about 39%, about 36.5% and 38.5%, about 36.5% and about 38%, about 36.5% and about 37.5%, about 36.5% and about 37%, about 37% and about 39%, about 37% and 38.5%, about 37% and about 38%, about 37% and about 37.5%, about 37.5% and about 39%, about 37.5% and 38.5%, about 37.5% and about 38%, about 38% and about 39%, about 38% and 38.5%, or about 38.5% and about 39% of the total lipid in the lipid nanoparticle.
  • the sterol is about 36%, about 36.5%, about 37%, about 37.5%, about 38%, about 38.5%, or about 39% of the total lipid in the lipid nanoparticle. In some embodiments, the sterol is at least 36%, at least 36.5%, at least 37%, at least 37.5%, at least 38%, at least 38.5%, or at least 39% of the total lipid in the lipid nanoparticle.
  • the conjugated lipid is between about 1.0% and about 4.0%, about 1.0% and about 3.5%, about 1.0% and about 3.0%, about 1.0% and about 2.5%, about 1.0% and about 2.0%, about 1.0% and about 1.5%, about 1.5% and about 4.0%, about 1.5% and about 3.5%, about 1.5% and about 3.0%, about 1.5% and about 2.5%, about 1.5% and about 2.0%, about 2.0% and about 4.0%, about 2.0% and about 3.5%, about 2.0% and about 3.0%, about 2.0% and about 2.5%, about 2.5% and about 4.0%, about 2.5% and about 3.5%, about 2.5% and about 3.0%, about 3.0% and about 4.0%, about 3.0% and about 3.5%, or about 3.5% and about 4.0% of the total lipid in the lipid nanoparticle.
  • the conjugated lipid is about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, or about 4.0% of the total lipid in the lipid nanoparticle. In some embodiments, the conjugated lipid is at least 1.0%, at least 1.5%, at least 2.0%, at least 2.5%, at least 3.0%, at least 3.5%, or at least 4.0% of the total lipid in the lipid nanoparticle.
  • the molar ratio of the cationic ionizable lipid, the helper lipid, the sterol, and the conjugated lipid is about 20%-80% : 5%-20% : 36%-39% : l%-4% (mol) of the total lipid present in the LNP composition.
  • the molar ratio of the cationic ionizable lipid, the helper lipid, the sterol, and the conjugated lipid is about 50% : 10% : 38.5% : 1.5% (mol) of the total lipid present in the LNP composition.
  • the cationic ionizable lipid is DLin-MC3-DMA
  • the helper lipid is DOPC
  • the sterol is cholesterol
  • the conjugated lipid is PEG-DMG
  • the molar ratio of the cationic ionizable lipid, the helper lipid, the sterol, and the conjugated lipid is about 50% : 10% : 38.5% : 1.5% (mol) of the total lipid present in the LNP composition.
  • the cationic ionizable lipid is DLin-KC2-DMA
  • the helper lipid is DOPC
  • the sterol is cholesterol
  • the conjugated lipid is PEG-DMG
  • the molar ratio of the cationic ionizable lipid, the helper lipid, the sterol, and the conjugated lipid is about 50% : 10% : 38.5% : 1.5% (mol) of the total lipid present in the LNP composition.
  • the composition comprises at least one lipid nanoparticle encapsulating the nucleic acid.
  • the lipid nanoparticle comprises a negative charge.
  • the lipid nanoparticle comprises a positive charge.
  • the terms “aqueous solution” and “buffer” are interchangeable.
  • the aqueous solution is a buffer selected from the group consisting of sodium acetate buffer, HEPES (4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid), PBS (phosphate buffered saline), TRIS (tris(hydroxymethyl)aminomethane), MES (2-(N- morpholino)ethanesulfonic acid), and citrate buffer.
  • the aqueous solution is sodium acetate buffer.
  • the aqueous solution is PBS.
  • the aqueous solution has a pH of about pH 5.0 to about pH 8.0. In some embodiments, the pH is about 5.0.
  • a lipid solution contains a mixture of lipids suitable to form lipid nanoparticles for encapsulation of nucleic acid (e.g., DNA or RNA).
  • a suitable lipid solution is ethanol based.
  • a suitable lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (i.e., 100% ethanol).
  • a suitable lipid solution is isopropyl alcohol based.
  • a suitable lipid solution is dimethylsulfoxide-based.
  • a suitable lipid solution is a mixture of suitable solvents including, but not limited to, ethanol, isopropyl alcohol and dimethylsulfoxide.
  • the nucleic acid is RNA. In some embodiments, the nucleic acid is DNA.
  • the N/P ratio is about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, about 5 to about 35, about 5 to about 30, about 5 to about 25, about 5 to about 20, about 5 to about 15, about 5 to about 10, about 10 to about 35, about 10 to about 30, about 10 to about 25, about 10 to about 20, about 10 to about 15, about 15 to about 35, about 15 to about 30, about 15 to about 25, about 15 to about 20, about 20 to about 35, about 20 to about 30, about 20 to about 25, about 25 to about 35, about 25 to about 30, or about 30 to about 35.
  • the nucleic acid is RNA and the N/P ratio is about 5 to about 30.
  • the nucleic acid is DNA and the N/P ratio is about 15 to about 20.
  • the nucleic acid is DNA and the N/P ratio is 4.
  • the term “zeta potential” refers to the electrostatic potential of a molecule or particle such as a LNP, measured at the plane of hydrodynamic slippage outside the surface of the molecule or particle. Usually measured by electrophoretic mobility. Related to the surface potential and a measure of the electrostatic forces of repulsion the particle is likely to meet when encountering another of the same sign of charge. In some embodiments, the zeta potential of the lipid nanoparticle, with or without the polymer coating, can be measured.
  • the LNP composition has a negative zeta potential.
  • the zeta potential is between about -0.1 mV to about -50 mV, about -0.5 mV to about -50 mV, about -1.0 mV to about -50 mV, about -2.0 mV to about -50 mV, about -5.0 mV to about -50 mV, about -10 mV to about -50 mV, about -15 mV to about -50 mV, about - 20 mV to about -50 mV, about -25 mV to about -50 mV, about -30 mV to about -50 mV, about -35 mV to about -50 mV, about -40 mV to about -50 mV, about -45 mV to about -50 mV, about -0.1 mV to about -45 mV, about -0.5 mV to about -45 mV, about -0.5 mV to
  • the zeta potential is at least -0.1 mV, at least -0.5 mV, at least -1.0 mV, at least -2.0 mV, at least -5.0 mV, at least -10 mV, at least -15 mV, at least -20 mV, at least -25 mV, at least -30 mV, at least -35 mV, at least -40 mV, at least -45 mV, or at least -50 mV.
  • the zeta potential is at most -0.1 mV, at most -0.5 mV, at most -1.0 mV, at most -2.0 mV, at most -5.0 mV, at most -10 mV, at most -15 mV, at most -20 mV, at most -25 mV, at most -30 mV, at most -35 mV, at most -40 mV, at most -45 mV, or at most -50 mV.
  • polydispersity index refers to the standard deviation (G) of the particle diameter distribution divided by the mean particle diameter.
  • G the standard deviation of the particle diameter distribution divided by the mean particle diameter.
  • PDI is used to estimate the average uniformity of a particle solution. For example, a small value, e.g., less than 0.3, indicates a narrow particle size distribution. For example, a larger PDI value corresponds to a larger size distribution in the particle sample.
  • the polydispersity index of the LNPs in a composition is less than 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1.
  • the polydispersity index of the LNPs in a composition is between about 0.1 to about 1.0, about 0.1 to about 0.9, about 0.1 to about 0.8, about 0.1 to about 0.7, about 0.1 to about 0.6, about 0.1 to about 0.5, about 0.1 to about 0.4, about 0.1 to about 0.3, about 0.1 to about 0.2, about 0.2 to about 1.0, about 0.2 to about 0.9, about 0.2 to about 0.8, about 0.2 to about 0.7, about 0.2 to about 0.6, about 0.2 to about 0.5, about 0.2 to about 0.4, about 0.2 to about 0.3, about 0.3 to about 1.0, about 0.3 to about 0.9, about 0.3 to about 0.8, about 0.3 to about 0.7, about 0.3 to about 0.6, about 0.3 to about 0.5, about 0.3 to about 0.4, about 0.4 to about 1.0, about 0.4 to about 0.9, about 0.4 to about 1.0, about 0.4 to about 0.9, about 0.4 to about 0.8, about 0.4 to about
  • the formed lipid nanoparticles are in an aqueous solution.
  • the aqueous solution is a buffer selected from the group consisting of sodium acetate buffer, HEPES (4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid), PBS (phosphate buffered saline), TRIS (tris(hydroxymethyl)aminomethane), MES (2-(N- morpholino)ethanesulfonic acid), and citrate buffer.
  • the aqueous solution is sodium acetate buffer.
  • the aqueous solution is PBS.
  • the LNP composition further comprises a sugar (e.g., the LNPs are in an aqueous solution comprising sugar).
  • the sugar is selected from the group consisting of lactose, sucrose, trehalose, and a combination thereof.
  • the amount of sugar in the LNP composition is between about 0% w/v and about 40% w/v. In one embodiment, the amount of sugar is between about 0.1% w/v and about 40% w/v, about 1% w/v to about 40% w/v, about 1% w/v to about 35% w/v, about 1% w/v to about 30% w/v, about 1% w/v to about 25% w/v, about 1% w/v to about 20% w/v, about 1% w/v to about 15% w/v, about 1% w/v to about 10% w/v, about 1% w/v to about 5% w/v, about 5% w/v to about 40% w/v, about 5% w/v to about 35% w/v, about 5% w/v to about 30% w/v, about 5% w/v to about 25% w/v, about 5% w/v to about 20% w/v.
  • therapeutic agent or “prophylactic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
  • Therapeutic agents are also referred to as “actives” or “active agents” or “active ingredients.” Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.
  • nucleic acid refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA, RNA, and hybrids thereof.
  • DNA may be in the form of antisense molecules, plasmid DNA, cDNA, PCR products, or vectors.
  • RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, dicer substrate RNA, circular RNA or viral RNA (vRNA), and combinations thereof.
  • Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).
  • PNAs peptide-nucleic acids
  • the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell.
  • Nucleotides contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
  • Bases include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.
  • a nucleic acid wherein the nucleic acid is DNA and at least 2 kilobases (kb), at least 3 kb, at least 4 kb, at least 5 kb, at least 5 kb, at least 7 kb, at least 8 kb, at least 9 kb, or at least 10 kb.
  • the nucleic acid molecule is between about 10 nucleotides (nt) and 20 kilobases (kb), about 500 nt to about 20 kb, about 1 kb to about 20 kb, about 2 kb to about 20 kb, about 3 kb to about 20 kb, about 4 kb to about 20 kb, about 5 kb to about 20 kb, about 6 kb to about 20 kb, about 7 kb to about 20 kb, about 8 kb to about 20 kb, about 9 kb to about 20 kb, about 10 kb to about 20 kb, about 15 kb to about 20 kb, about 10 nt and 15 kb, about 500 nt to about 15 kb, about 1 kb to about 15 kb, about 2 kb to about 15 kb, about 3 kb to about 15 kb, about 4 kb to about 15 kb, about 5 kb
  • the nucleic acid is DNA, e.g., viral vector or plasmid, and is about 3 kb to about 20 kb.
  • the nucleic acid is DNA and at least 2 kilobases (kb), at least 3 kb, at least 4 kb, at least 5 kb, at least 5 kb, at least 7 kb, at least 8 kb, at least 9 kb, or at least 10 kb.
  • the nucleotide analog may be any molecule obtained by modifying a ribonucleotide, deoxyribonucleotide, RNA, or DNA to improve nuclease resistance, to stabilize, to enhance affinity with a complementary-strand nucleic acid or to enhance cell permeability as compared with RNA or DNA, or for visualization.
  • the nucleotide analog may be a naturally- occurring molecule or a non-natural molecule, and examples thereof include a sugar-modified nucleotide analog and a phosphodiester bond-modified nucleotide analog.
  • the sugar-modified nucleotide analog may be any one obtained by addition of or substitution with a substance having an arbitrary chemical structure for a part or the whole of the chemical structure of the sugar of a nucleotide. Specific examples thereof include a nucleotide analog substituted with 2'-O-methylribose, a nucleotide analog substituted with 2'- O-propylribose, a nucleotide analog substituted with 2'-methoxy ethoxyribose, a nucleotide analog substituted with 2'-O-methoxyethylribose, a nucleotide analog substituted with 2'-O- [2-(guanidinium)ethyl]ribose, a nucleotide analog substituted with 2'-O-fluororibose, a crosslinked artificial nucleic acid provided with two cyclic structures by introducing a crosslinked structure to the sugar moiety (Bridged Nucle
  • the phosphodiester bond-modified nucleotide analog may be any one obtained by addition of or substitution with an arbitrary chemical substance for a part or the whole of the chemical structure of the phosphodiester bond of a nucleotide. Specific examples thereof include a nucleotide analog substituted with a phosphorothioate bond and a nucleotide analog substituted with an N3'-P5' phosphoramidate bond.
  • the nucleic acid derivative may be any molecule obtained by adding to a nucleic acid another chemical substance to improve nuclease resistance, to stabilize, to enhance affinity with a complementary-strand nucleic acid, or to enhance cell permeability as compared with the nucleic acid, or for visualization.
  • Specific examples thereof include a derivative with 5'- polyamine added, a derivative with cholesterol added, a derivative with steroid added, a derivative with bile acid added, a derivative with vitamin added, a derivative with Cy5 added, a derivative with Cy3 added, a derivative with 6-FAM added, and a derivative with biotin added.
  • the nucleic acid in the present invention is not limited to a particular nucleic acid, and may be a nucleic acid, for example, for the purpose of ameliorating a disease, a symptom, a disorder, or sickness, and mitigating a disease, a symptom, a disorder, or pathological condition or preventing the onset thereof (herein, occasionally referred to as “treatment or the like of a disease”), or a nucleic acid for regulating expression of a desired protein that is useful for research, even though the protein does not contribute to treatment or the like of a disease.
  • nucleic acid in the present invention examples include siRNAs, shRNAs, miRNAs, miRNA mimics, antisense nucleic acids, ribozymes, mRNAs, decoy nucleic acids, aptamers, plasmid DNAs, Cosmid DNAs, and BAC DNAs.
  • the nucleic acid is preferably an RNA such as an siRNA and an mRNA or an analog or derivative obtained by artificially modifying an RNA.
  • the “siRNA” refers to a double-stranded RNA or relative thereof consisting of 10 to 30 nucleotides, preferably of 15 to 25 nucleotides, and including complementary sequences.
  • the siRNA includes protruding nucleotides preferably of one to three nucleotides, more preferably of two nucleotides, at the 3 '-end.
  • the moiety of complementary sequences may be completely complementary or include non-complementary nucleotides, but preferably are completely complementary.
  • the siRNA in the present invention is not limited to a particular siRNA, and, for example, an siRNA for knockdown of gene expression against a disease-related gene may be used.
  • a disease-related gene refers to any gene or polynucleotide generating a transcription or translation product at an abnormal level or in an abnormal form in cells derived from tissue of a patient, as compared with tissue or cells from a disease-free control.
  • an siRNA to regulate expression of a desired protein useful for research may be used.
  • the “mRNA” refers to an RNA including a nucleotide sequence that can be translated into a protein.
  • the mRNA in the present invention is not limited to a particular mRNA and may be any mRNA capable of expressing a desired protein in cells.
  • the mRNA is preferably an mRNA useful for pharmaceutical applications (e.g., applications of disease treatment) and/or applications for research, and examples of such mRNAs include an mRNA to express a marker protein such as luciferase in cells.
  • gene refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide or precursor polypeptide.
  • Gene product refers to a product of a gene such as an RNA transcript or a polypeptide.
  • RNA means a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide is meant a nucleotide with a hydroxyl group at the 2' position of a P ⁇ D ⁇ ribo-furanose moiety.
  • the terms includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of an interfering RNA or internally, for example at one or more nucleotides of the RNA.
  • Nucleotides in the RNA molecules of the instant disclosure can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
  • ribonucleic acid and “RNA” refer to a molecule containing at least one ribonucleotide residue, including siRNA, antisense RNA, single stranded RNA, microRNA, mRNA, noncoding RNA, and multivalent RNA.
  • the RNA is a self-replicating RNA.
  • the RNA is mRNA.
  • the RNA is siRNA.
  • the nucleic acid is from about 1000 nucleotides to about 13000 nucleotides in length.
  • the lipid nanoparticles of the disclosure also typically have a total lipidmucleic acid (e.g., DNA or RNA) ratio (mass/mass ratio) of from about 1:1 to about 100:1, from about 1:1 to about 50:1, from about 2:1 to about 45:1, from about 3:1 to about 40:1, from about 5:1 to about 38:1, or from about 6:1 to about 40:1, or from about 7:1 to about 35:1, or from about 8:1 to about 30:1; or from about 10:1 to about 25:1; or from about 8:1 to about 12:1; or from about 13:1 to about 17:1; or from about 18:1 to about 24: 1; or from about 20:1 to about 30:1.
  • a total lipidmucleic acid (e.g., DNA or RNA) ratio (mass/mass ratio) of from about 1:1 to about 100:1, from about 1:1 to about 50:1, from about 2:1 to about 45:1, from about 3:1 to about 40:1, from about 5:1 to about 38:1,
  • the lipid nanoparticles comprise a nucleic acid (e.g., DNA or RNA) that is fully encapsulated within the lipid portion of the formulation, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about
  • the encapsulated nucleic acid is calculated by the formula: [(total nucleic molcculcs frcc nucleic molecules)/(total nucleic molecules)] x 100%.
  • RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.
  • the disease is not limited to a particular disease, and examples of the disease include diseases listed below. Contents in each “( )” are examples of the corresponding disease- related gene, except for the case that specific disease examples are listed.
  • Another example of the nucleic acid in the present invention is a nucleic acid that regulates the expression level of any of those disease-related genes (or a protein encoded by the disease-related gene).
  • ALS SOD1, ALS2, STEX, FUS, TARDBP, VEGF
  • Alzheimer's disease APP, AAA, CVAP, ADI, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP, A2M, BLMH, BMH, PSEN1, AD3), autism (BZRAP1, MDGA2, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2), fragile X syndrome (FMR2, FXR1, FXR2, mGLUR5), Huntington's disease (HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17), Parkinson's disease (NR4A2, NURR1, NOT, TINUR, SNCAIP,
  • eye diseases [age-related macular degeneration (Aber, Ccl2, cp, Timp3, cathepsin D, Vldlr, Ccr2), cataract (CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, PAX6, AN2, MGDA, CRYBAI, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BSFP2, CP49, CP47, HSF4, CTM, MW, AQPO, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1), corneal opacity (APOA1, TGFB1, CSD2, CDGG1, CSD, BIGH3, CDG2, TASTD2, TROP2, M1S1, VS, corneal o
  • neoplastic diseases malignant tumor, neovascular glaucoma, infantile hemangioma, multiple myeloma, chronic sarcoma, metastatic melanoma, Kaposi's sarcoma, angiopro liferation, cachexia, metastasis or the like of breast cancer, cancer (e.g., colorectal cancer (e.g., familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumor), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (e.g., pancreatic ductal carcinoma), gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous cell carcinoma), breast cancer (e.g., invasive ductal breast carcinoma
  • composition of the present invention as a drug may be produced by using a method as disclosed herein with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier examples include formulations for oral administration (e.g., a capsule, tablet, or liquid) blended with a conventional auxiliary such as a buffering agent and/or a stabilizer.
  • examples of the dosage form of the drug include formulations for parenteral administration (e.g., a liquid such an injection) blended with a conventional auxiliary such as a buffering agent and/or a stabilizer.
  • parenteral administration e.g., a liquid such an injection
  • a conventional auxiliary such as a buffering agent and/or a stabilizer
  • examples of the dosage form of the drug include formulations for topical administration, such as an ointment, a cream, a liquid, and a plaster, blended with a conventional pharmaceutical carrier.
  • the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the phrase “pharmaceutically acceptable excipient,” refers to any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient.
  • Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration.
  • antiadherents antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration.
  • excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C,
  • the composition of the present invention may be used for introduction of an active ingredient into various types of cells, tissues, or organs.
  • cells to which the composition of the present invention may be applied include mesenchymal stem cells, neural stem cells, skin stem cells, splenocytes, nerve cells, glial cells, pancreatic B cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, myoblasts, muscle satellite cells, smooth muscle cells), fat cells, blood cells (e.g., macrophages, T cells, B cells, natural killer cells, mast cells, leukocytes, neutrophils, basophils, eosinophils, monocytes, megakaryocytes, hematopoietic stem cells), synoviocytes, chondrocytes, osteocytes, osteoblasts, osteoclasts,
  • tissues or organs to which the composition of the present invention may be applied include all tissues or organs in which the above cells are present, for example, brain, sites of brain (e.g., olfactory bulb, amygdala, basal ganglion, hippocampus, thalamus, hypothalamus, subthalamic nucleus, cerebral cortex, medulla oblongata, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, callosum, substantia nigra), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, digestive tract (e.g., large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, placenta, uterus, bones
  • composition of the present invention is particularly superior in efficiency to introduce a nucleic acid into target cells.
  • the compound, the lipid particle, and the composition of the present invention are stable and have low toxicity and can be safely used.
  • the composition of the present invention in vivo, or using the composition as a drug, the composition is suitably administered to a subject (e.g., a human or a non-human mammal (preferably, a human)) so that an effective amount of the nucleic acid can be delivered to targeted cells.
  • a subject e.g., a human or a non-human mammal (preferably, a human)
  • the composition in vivo, or using the composition as a drug, can be orally or parenterally (e.g., local, rectal, or intravenous administration) administered in a safe manner in the form of a pharmaceutical formulation such as tablets (including sugar-coated tablets, fdm-coated tablets, sublingual tablets, orally disintegrating tablets), powders, granules, capsules (including soft capsules, microcapsules), liquids, troches, syrups, emulsions, suspensions, injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), topical formulations (e.g., formulations for nasal administration, transdermal formulations, ointments), suppositories (e.g., rectal suppositories, vaginal suppositories), pellets, nasal formulations, pulmonary formulations (inhalations), and infusions.
  • a pharmaceutical formulation such as tablets (including sugar-coated tablets,
  • an “effective amount” or “therapeutically effective amount” of an active agent or therapeutic agent such as a therapeutic nucleic acid is an amount sufficient to produce the desired effect, e.g. an increase or inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of the nucleic acid.
  • An increase in expression of a target sequence is achieved when any measurable level is detected in the case of an expression product that is not present in the absence of the nucleic acid.
  • an in increase in expression is achieved when the fold increase in value obtained with a nucleic acid such as siRNA relative to control is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or greater.
  • Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as antisense oligonucleotide relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%.
  • Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of suitable reporter proteins, as well as phenotypic assays known to those of skill in the art.
  • composition of the present invention provides methods of producing a pharmaceutical composition, such as lipid nanoparticles, as disclosed herein.
  • the method of producing a pharmaceutical composition comprising a. producing lipid nanoparticle (LNP) by mixing a lipid mixture and an aqueous solution, wherein the lipid mixture comprises: i.
  • a cationic ionizable lipid wherein the cationic ionizable lipid is (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4- (Dimethylamino)butanoate (DLin-MC3-DMA), or 2-(2,2-Di((9Z,12Z)-octadeca-9,12-dien-l- yl)-l,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA); ii.
  • DLin-MC3-DMA 6-(2,2-Di((9Z,12Z)-octadeca-9,12-dien-l- yl)-l,3-dioxolan-4-yl)-N,N-dimethylethanamine
  • helper lipid wherein the helper lipid is selected from the group consisting of l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), and 1 ,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC); iii.
  • DSPC l,2-distearoyl-sn-glycero-3- phosphocholine
  • DOPE 1,2- dioleoyl-sn-glycero-3-phosphocholine
  • DOPC 1,2- dioleoyl-sn-glycero-3-phosphocholine
  • DMPC 1,2- dioleoyl-
  • a sterol wherein the sterol is selected from the group consisting of cholesterol, P-sitosterol, campesterol, ergosterol, phytosterols (e.g., stigmastanol), and fucosterol;
  • a conjugated lipid wherein the conjugated lipid is selected from the group consisting of 1 ,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG)-IOOOK, PEG-DMG-2000K, PEG-DMG-3000K, PEG-DMG-4000K, and PEG-DMG-5000K; and the aqueous solution comprises: iv.
  • nucleic acid wherein the nucleic acid is DNA and at least 2 kilobases (kb), at least 3 kb, at least 4 kb, at least 5 kb, at least 5 kb, at least 7 kb, at least 8 kb, at least 9 kb, or at least 10 kb; and b. mixing the LNPs produced in step a) and a pharmaceutically acceptable carrier.
  • kb kilobases
  • the aqueous solution is a buffer selected from the group consisting of sodium acetate buffer, HEPES (4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid), PBS (phosphate buffered saline), TRIS (tris(hydroxymethyl)aminomethane), MES (2- (N-morpholino)ethanesulfonic acid), and citrate buffer.
  • the aqueous solution is sodium acetate buffer.
  • the aqueous solution is PBS.
  • a lipid solution contains a mixture of lipids suitable to form lipid nanoparticles for encapsulation of nucleic acid (e.g., DNA or RNA).
  • a suitable lipid solution is ethanol based.
  • a suitable lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (i.e., 100% ethanol).
  • a suitable lipid solution is isopropyl alcohol based.
  • a suitable lipid solution is dimethylsulfoxide-based.
  • a suitable lipid solution is a mixture of suitable solvents including, but not limited to, ethanol, isopropyl alcohol and dimethylsulfoxide.
  • the nucleic acid molecule is DNA or RNA. In some embodiments, the nucleic acid molecule is DNA.
  • the LNP composition further comprises a sugar (e.g., the LNPs are in an aqueous solution comprising sugar).
  • the sugar is selected from the group consisting of lactose, sucrose, trehalose, and a combination thereof.
  • the amount of sugar in the LNP composition is between about 0% w/v and about 40% w/v. In one embodiment, the amount of sugar is between about 0.1% w/v and about 40% w/v, about 1% w/v to about 40% w/v, about 1% w/v to about 35% w/v, about 1% w/v to about 30% w/v, about 1% w/v to about 25% w/v, about 1% w/v to about 20% w/v, about 1% w/v to about 15% w/v, about 1% w/v to about 10% w/v, about 1% w/v to about 5% w/v, about 5% w/v to about 40% w/v, about 5% w/v to about 35% w/v, about 5% w/v to about 30% w/v, about 5% w/v to about 25% w/v, about 5% w/v to about 20% w/v.
  • the amount of sugar is at least 5% w/v, at least 10% w/v, at least 20% w/v, or at least 30% w/v. In one embodiment, preferably the amount of sugar is about 5% w/v. Lyophilization
  • the pharmaceutical composition is lyophilized and reconstituted for administration to a subject.
  • the lyophilized pharmaceutical composition that, when reconstituted, has a minimal amount of large aggregates.
  • Such large aggregates may have a size greater than about 0.2 pm, greater than about 0.5 pm, or greater than about 1 pm, and can be undesirable in a reconstituted solution.
  • Aggregate sizes can be measured using a variety of techniques including those indicated in the U.S. Pharmacopeia 32 ⁇ 788>, hereby incorporated by reference.
  • the tests may include a light obscuration particle count test, microscopic particle count test, laser diffraction, and single particle optical sensing.
  • the particle size in a given sample is measured using laser diffraction and/or single particle optical sensing.
  • DLS may be used to measure particle size, but it relies on Brownian motion so the technique may not detect some larger particles.
  • Laser diffraction relies on differences in the index of refraction between the particle and the suspension media.
  • the technique is capable of detecting particles at the submicron to millimeter range. Relatively small (e.g., about 1-5 wt %) amounts of larger particles can be determined in nanoparticle suspensions.
  • Single particle optical sensing (SPOS) uses light obscuration of dilute suspensions to count individual particles of about 0.5 pm. By knowing the particle concentration of the measured sample, the weight percentage of aggregates or the aggregate concentration (particles/mL) can be calculated.
  • Formation of aggregates can occur during the freezing and/or drying steps of lyophilization, e.g., due to the dehydration of the surface of the particles.
  • the freezing process has a concentrating effect that can reduce the distance between the particles as the ice forms (Alison et al., Biochim Biophys Acta. 2000 Sep. 29; 1468(1-2): 127-38; Armstrong and Anchordoquy, J Pharm Sci. 2004 November; 93(11):2698-709).
  • This dehydration can be avoided by using lyoprotectants, such as polysaccharides, in the suspension before lyophilization.
  • Suitable polysaccharides include sucrose, lactulose, lactose, maltose, trehalose, or cellobiose, kojibiose, nigerose, isomaltose, trehalose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobiase, melibiose, melibiulose, rutinose, rutinulose, and xylobiose.
  • the composition comprises a polysaccharide that is sucrose.
  • the composition comprises a polysaccharide that is trehalose.
  • the invention provides a method of preventing substantial aggregation of particles in a pharmaceutical nanoparticle composition comprising adding a sugar and a salt to the lyophilized formulation to prevent aggregation and release of nucleic acid from the interior of the liposome
  • the lyophilized pharmaceutical composition comprising LNPs can be reconstituted in an appropriate buffers, for example, but not limited to, water, PBS, and HEPES.
  • the lyophilized pharmaceutical composition comprising LNPs is reconstituted in buffer at a pH between about 5.0 and 8.0. In some embodiments, the pH is 7.4.
  • a device is used to administer the formulations to the lungs.
  • Suitable devices include, but are not limited to, dry powder inhalers, pressurized metered dose inhalers, nebulizers, and electrohydrodynamic aerosol devices.
  • the liquid formulations comprising the LNPs described above can also be administered using a nebulizer.
  • Nebulizers are liquid aerosol generators that convert the liquid formulation described able, usually aqueous-based compositions, into mists or clouds of small droplets, preferably having diameters less than 5 microns mass median aerodynamic diameter, which can be inhaled into the lower respiratory tract. This process is called atomization.
  • the droplets carry the one or more active agents into the nose, upper airways or deep lungs when the aerosol cloud is inhaled.
  • Any type of nebulizer may be used to administer the formulation to a patient, including, but not limited to pneumatic (jet) nebulizers and electromechanical nebulizers.
  • the present invention provides methods of treating a subject by administering the composition as disclosed herein.
  • the lyophilized composition, as disclosed herein can be administered orally, via nebulization, intravenously, subcutaneously, intramuscularly, intraperitoneally, topically, and by infusions.
  • patient refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.
  • subject refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes.
  • Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans).
  • treating refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition.
  • “treating” cancer may refer to inhibiting survival, growth, and/or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • the present invention provides methods of optimizing a Lipid Nanoparticle (LNP) comprising steps of (1) screening LNPs in vitro and (2) screening LNPs in vivo using immune deficient animal.
  • LNP Lipid Nanoparticle
  • the LNP can be optimized from many perspectives including the type of each component, the ratio of the components, the payload encapsulated in the LNP and the N/P ratio.
  • LNPs are tested in vitro for transfection efficiency including protein expression followed by an evaluation in the in vivo model using immune deficient animal in the method.
  • Subject LNPs in the in vitro test can be screened to limited number of LNPs and the screened LNPs can be evaluated in the immune deficient animal in vivo model.
  • the parameters to be assessed in the immune deficient animal in vivo model may be selected appropriately by the person skilled in the art.
  • the parameters may include protein expression, payload (e.g., nucleic acid) delivery or distribution, durability of expression, dose titration and toxicity, including immunotoxicity.
  • the physicochemical characteristics of LNPs are tested followed by the in vitro testing for transfection efficiency.
  • the physicochemical characteristics may be selected appropriately by the person skilled in the art.
  • the physicochemical characteristics may include the size, charge, N/P ratio and encapsulation efficiency.
  • the parameters may include protein expression and toxicity including immunotoxicity.
  • Subject LNPs can be screened to a reduced number of LNPs through the physicochemical characteristics testing and the screened LNPs can be evaluated in the subsequent in vitro testing or in vivo testing.
  • the LNPs are evaluated in an in vivo model using wild type animal following the immune deficient animal in vivo model evaluation.
  • the parameters to be assessed in the wild type animal in vivo model may be selected appropriately by the person skilled in the art.
  • the parameters may include protein expression, payload (e.g., nucleic acid) delivery or distribution, durability of expression, dose titration and toxicity, including immunotoxicity.
  • the LNPs are evaluated in an in vivo model using animal having a disease of interest following the wild type animal in vivo model evaluation.
  • the parameters to be assessed in the disease animal in vivo model may be selected appropriately by the person skilled in the art based on the disease of interest.
  • the parameters may include protein expression and any pharmacodynamic parameters that may enable evaluation of the therapeutic effect by the payload in the LNP.
  • any part of or whole steps may be repeated to screen LNPs.
  • the components, ratio of the components, payload of the LNPs may be varied to screen and adjust them.
  • the animal used in the method is selected appropriately by the person skilled in the art.
  • the animal may be a mouse, rat, rabbit, dog, porcine and/or monkey.
  • an immune deficient animal a person skilled in the art can select appropriately from animals with lack of or deficient immune response against the exogenous molecules.
  • the immune deficient animal may be NOD animal, NSG animal, SCID animal, or an animal deficient in specific components of the immune and/or DNA sensing pathways including cGAS knockout animal, STING knockout animal or an animal with knockout elements of the interferon signaling pathway.
  • NOD mouse, NSG mouse or SCID mouse is preferable. Examples
  • lonizable-lipids, helper lipids, cholesterol and PEG-DMG were dissolved in ethanol to make 25 mM stock of each respectively which were than mixed at a molar ratio of 50/10/38.5/1.5.
  • Nucleic acid of interest was diluted in 25 mM sodium acetate buffer (pH 5).
  • the lipid mixture and nucleic acid solution were mixed using a microfluidic micromixer (Precision Nanosystems, Vancouver, BC, Canada) at room temperature with a flow rate ratio of 3 ml/min:9 mL/min to afford a dispersion containing lipid nanoparticles (LNPs) encapsulating respective nucleic acid.
  • LNPs lipid nanoparticles
  • LNPs obtained were either dialyzed or filtered through Amicon U-15 filters (30 kDa or 100 kDa, cellulose) tube at 2000 x g 4 °C for 30 min to remove excess ethanol and replace buffer with PBS. Sterile sucrose solution (20% final volume) was added to the formulations as cryoprotectant. Subsequently, formulations were filtered through 0.2 pm syringe filter. Quant-iT TM PicoGreen dsDNA assay kit (Thermo Fisher Scientific) was used as manufacturer’s protocol to quantify amount of nucleic acid encapsulated in the LNPs.
  • the particle size and PDI of the LNPs were measured by using a Zetasizer Ultra Zs Xplorer (Malvern Instruments Limited). The formulations were kept at -80 °C until used for in vitro / in vivo experiments.
  • N/P ratio of LNP formulations including amino lipids and DSPC were optimal at approximately 4-6 and resulted in mild expression.
  • Lipid 1-DSPC resulted with severe toxicity.
  • MC3-DOPC and MC3-DOPE resulted in surprising increased protein expression at a N/P ratio of 4. Moderate toxicity was seen with MC3-DSPC formulation.
  • Lipid 1-DOPC consistently produced high levels of hATP7B and eGFP. Codon optimized hATP7B was also determined to result in surprisingly higher expression than wild type sequence.
  • Table 2 shows LNP formulations and physical properties as shown in FIGs. 1A-1C. Ionizable lipids, helper lipids, sterol, and PEG2K-DMG were at a lipid molar ratio of 50%:10%:38.5%:1.5%.
  • FIG. 2A shows percentage of viable cells as function of N/P ratio following incubation with LNP-npDNA(CAG-Luc) containing MC3 as ionizable-lipid.
  • FIG. 2B shows luciferase expression as function of N/P ratio following incubation with LNP-npDNA(CAG- Luc) containing MC3 as ionizable-lipid.
  • FIG. 3 A shows the percentage of viable cells as function of N/P ratio following incubation with LNP-npDNA(ATP7B) containing ionizable-lipids shown in legend.
  • FIGs. 3B and 3C DNA expression as function of N/P ratio following incubation with LNP-npDNA(ATP7B) containing ionizable-lipids shown in legend.
  • Treatment of Huh7 cells were performed using 50 pg/mL stock instead of 25 pg/mL stock for other groups.
  • Table 3 shows LNP formulations and physical properties screening helper lipids. Ionizable lipids, helper lipids, sterol, and PEG2K-DMG were at a lipid molar ratio of 50%: 10%:38.5%: 1.5%.
  • FIG. 4 A shows LNP- npDNA(ATP7B) physio-chemical properties, including particle size and PDI of LNP formulations.
  • FIG. 4B shows dependence of particle size (bar) and encapsulation efficiency (star symbol) on N/P ratio and helper lipids for LNP formulations.
  • FIG. 4C shows cell viability as function of on N/P ratio and helper lipids for LNP formulations.
  • Table 4 shows helper lipid screening as shown in FIG. 5.
  • FIG. 6 A shows cell viability dose response for Lipid 1 Ionizable Lipid-DSPC.
  • FIG. 6B shows the impact of LNP-npDNA(CAG-Luc) dose on transgene expression.
  • Table 5 shows amino lipid comparison with optimal helper lipid and N/P ratio. Ionizable lipids, helper lipids, sterol, and PEG2K-DMG were at a lipid molar ratio of 50%:10%:38.5%:1.5%.
  • Table 6 shows amino lipid comparison with optimal helper lipid and N/P combination.
  • LNP-DNA vectors with DOPC helper lipid component were generally well tolerated even at 1 mg/kg.
  • Lipid 1-DOPC formulation was the only successful formulation in achieving in-vivo protein expression of hATP7B which was however 4 fold lower than that measured in human liver. Corresponding mRNA and DNA levels measured in this group was significantly higher compared to nearly background levels measured in MC3 and KC2 containing formulations. [0179] In parallel, the co-dosed reporter formulation of Lipid 1-DOPC with eGFP nanoplasmid payload also outperformed the MC3 and KC2 achieving about 40 fold higher expression than a MC3 lipid formulation. (FIGs. 9A-9F). [0180] Table 7 shows comparative evaluation of multiple transgene expression in vivo utilizing LNP-DNA formulations. Ionizable lipids, helper lipids, sterol, and PEG2K-DMG were at a lipid molar ratio of 50%: 10%:38.5%: 1.5%.
  • DNA constructs used include:
  • Table 8 shows TA dosing in immune deficient mice (6-8 weeks old male mice).
  • mice purchased from Jackson Laboratories were dosed intravenously at indicated dose levels at volume of 10ml per kg. Terminal liver collections were performed at Day 7 post dose for liver resident proteins and Day 28 for secreted proteins.
  • Western blotting, ELISA and immunohistochemistry based protein measurements were used to quantify transgene product expression.
  • mRNA and DNA quantification was performed using ddPCR to obtain copy numbers/pg nucleic acid or genomic DNA respectively.
  • FVIII and GLA protein activity was determined using pre-established assays .
  • Protein measurement for hATP7B, eGFP and hOTC was performed by specific ELISA assays and substantial protein was detected in mouse livers compared to saline treated controls.
  • npDNA was found resident in liver tissue at Day 7 ( ⁇ le6 copies/pg genomic DNA) while lower levels ( ⁇ le4 copies/pg gDNA) was observed in the Day 28 samples.
  • mRNA expressed was positively detected by ddPCR between (le3 and le5 copies /pg nucleic acid) for the different payload constructs as seen in the figure and has a correlation to the size, sequence, promoter used and elapsed time post dose. Larger sized transgenes like ATP7B and FVIII have lesser copies than smaller transgenes like PAH and OTC. For FVIII constructs, the ApoE-AAT promoter had much higher expression compared to CAG promoter. Similarly, Day 28 tissues on average have ⁇ 10 fold reduction in mRNA copies although direct comparison of time points was not performed for the same payload.
  • Substantial protein expression was detected in all payloads corresponding to the presence of payload DNA and mRNA expressed. Presence of liver resident proteins OTC, PAH and eGFP was visible in hepatocytes of liver sections as seen by IHC. Activity was secreted proteins FVIII and GLA was detected in serum from Day 7 through Day 28: FVIII activity decreased from 20% at Day 7 to undetected by Day 28 in the CAG promoter construct versus was more stable between 20%-30% for the ApoE-AAT promoter in the same duration. GLA activity on the other hand increased from lOOnM/hr/mL to 300nM/hr/mL going from Day 7 to Day 28 owing to its better serum stability. Levels of protein expression for hATP7B varied substantially from 36%-128% of human liver levels of hATP7B. (FIGs.
  • Table 9 shows promoter and transgene constructs.
  • GLA activity measured was 60nM/hr/mL of serum collected at Day 7 in wt mice treated with saline (“control”) and with LNP-DNA GLA-OTC (dose: 1.6 mg/kg). GLA activity was 2-3 fold lower than levels observed in immune deficient mice.
  • FIG. 15C [0200] hFVIII activity similarly trended lower in WT mice (dose: 2 mg/kg) compared to immune deficient mice (dose: 1.4 mg/kg) and was only measured around 2-11% of normal human level.
  • FIG. 15D

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Abstract

L'invention concerne de nouvelles formulations de nanoparticules lipidiques et leur utilisation. Les nanoparticules lipidiques comprennent des lipides ionisables cationiques, des lipides auxiliaires, des stérols et des lipides conjugués. Les nanoparticules lipidiques comprenant en outre des agents thérapeutiques et/ou prophylactiques tels que l'ADN sont utiles dans l'administration d'agents thérapeutiques et/ou prophylactiques à des cellules ou à des organes (par exemple, des cellules ou des organes de mammifère) pour, par exemple, réguler l'expression de polypeptides, de protéines ou de gènes.
PCT/IB2024/000233 2023-05-15 2024-05-15 Compositions et procédés d'administration d'acides nucléiques à des cellules Pending WO2024236361A1 (fr)

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Citations (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010042877A1 (fr) 2008-10-09 2010-04-15 Tekmira Pharmaceuticals Corporation Lipides aminés améliorés et procédés d'administration d'acides nucléiques
WO2010054401A1 (fr) 2008-11-10 2010-05-14 Alnylam Pharmaceuticals, Inc. Nouveaux lipides et nouvelles compositions pour l’administration d’agents thérapeutiques
WO2011153493A2 (fr) 2010-06-03 2011-12-08 Alnylam Pharmaceuticals, Inc. Lipides biodégradables pour l'administration de principes actifs
WO2013126803A1 (fr) 2012-02-24 2013-08-29 Protiva Biotherapeutics Inc. Lipides cationiques trialkylés et leurs procédés d'utilisation
WO2014007398A1 (fr) 2012-07-06 2014-01-09 協和発酵キリン株式会社 Lipide cationique
WO2015005253A1 (fr) 2013-07-08 2015-01-15 第一三共株式会社 Nouveau lipide
WO2015011633A1 (fr) 2013-07-23 2015-01-29 Protiva Biotherapeutics, Inc. Compositions et procédés pour l'administration d'arn messager
US20150239834A1 (en) 2013-11-18 2015-08-27 Arcturus Therapeutics, Inc. Symmetric ionizable cationic lipid for rna delivery
WO2015199952A1 (fr) 2014-06-25 2015-12-30 Acuitas Therapeutics Inc. Nouveaux lipides et formulations nanoparticulaires lipidiques pour l'administration d'acides nucléiques
WO2016021683A1 (fr) 2014-08-07 2016-02-11 武田薬品工業株式会社 Lipide cationique
WO2016104580A1 (fr) 2014-12-26 2016-06-30 エーザイ・アール・アンド・ディー・マネジメント株式会社 Lipide cationique
WO2017000414A1 (fr) 2015-06-30 2017-01-05 宇龙计算机通信科技(深圳)有限公司 Procédé de détection de service, système de détection de service, terminal et station de base
WO2017075531A1 (fr) 2015-10-28 2017-05-04 Acuitas Therapeutics, Inc. Nouveaux lipides et nouvelles formulations de nanoparticules de lipides pour l'administration d'acides nucléiques
WO2017117528A1 (fr) 2015-12-30 2017-07-06 Acuitas Therapeutics, Inc. Lipides et formulations de nanoparticules de lipides pour la libération d'acides nucléiques
WO2019131839A1 (fr) 2017-12-28 2019-07-04 武田薬品工業株式会社 Lipides cationiques
WO2020032184A1 (fr) 2018-08-10 2020-02-13 武田薬品工業株式会社 Lipide cationique
US20200315967A1 (en) * 2016-06-24 2020-10-08 Modernatx, Inc. Lipid nanoparticles
CN114480442A (zh) * 2020-10-27 2022-05-13 深圳吉诺因生物科技有限公司 一种mRNA及包含其的新型冠状病毒mRNA疫苗
WO2022251959A1 (fr) * 2021-06-01 2022-12-08 Nanovation Therapeutics Inc. Administration de vecteur d'adn à l'aide de nanoparticules lipidiques

Patent Citations (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010042877A1 (fr) 2008-10-09 2010-04-15 Tekmira Pharmaceuticals Corporation Lipides aminés améliorés et procédés d'administration d'acides nucléiques
WO2010054401A1 (fr) 2008-11-10 2010-05-14 Alnylam Pharmaceuticals, Inc. Nouveaux lipides et nouvelles compositions pour l’administration d’agents thérapeutiques
WO2011153493A2 (fr) 2010-06-03 2011-12-08 Alnylam Pharmaceuticals, Inc. Lipides biodégradables pour l'administration de principes actifs
WO2013126803A1 (fr) 2012-02-24 2013-08-29 Protiva Biotherapeutics Inc. Lipides cationiques trialkylés et leurs procédés d'utilisation
WO2014007398A1 (fr) 2012-07-06 2014-01-09 協和発酵キリン株式会社 Lipide cationique
WO2015005253A1 (fr) 2013-07-08 2015-01-15 第一三共株式会社 Nouveau lipide
WO2015011633A1 (fr) 2013-07-23 2015-01-29 Protiva Biotherapeutics, Inc. Compositions et procédés pour l'administration d'arn messager
US20150239834A1 (en) 2013-11-18 2015-08-27 Arcturus Therapeutics, Inc. Symmetric ionizable cationic lipid for rna delivery
WO2015199952A1 (fr) 2014-06-25 2015-12-30 Acuitas Therapeutics Inc. Nouveaux lipides et formulations nanoparticulaires lipidiques pour l'administration d'acides nucléiques
WO2016021683A1 (fr) 2014-08-07 2016-02-11 武田薬品工業株式会社 Lipide cationique
WO2016104580A1 (fr) 2014-12-26 2016-06-30 エーザイ・アール・アンド・ディー・マネジメント株式会社 Lipide cationique
WO2017000414A1 (fr) 2015-06-30 2017-01-05 宇龙计算机通信科技(深圳)有限公司 Procédé de détection de service, système de détection de service, terminal et station de base
WO2017075531A1 (fr) 2015-10-28 2017-05-04 Acuitas Therapeutics, Inc. Nouveaux lipides et nouvelles formulations de nanoparticules de lipides pour l'administration d'acides nucléiques
WO2017117528A1 (fr) 2015-12-30 2017-07-06 Acuitas Therapeutics, Inc. Lipides et formulations de nanoparticules de lipides pour la libération d'acides nucléiques
US20200315967A1 (en) * 2016-06-24 2020-10-08 Modernatx, Inc. Lipid nanoparticles
WO2019131839A1 (fr) 2017-12-28 2019-07-04 武田薬品工業株式会社 Lipides cationiques
WO2020032184A1 (fr) 2018-08-10 2020-02-13 武田薬品工業株式会社 Lipide cationique
CN114480442A (zh) * 2020-10-27 2022-05-13 深圳吉诺因生物科技有限公司 一种mRNA及包含其的新型冠状病毒mRNA疫苗
WO2022251959A1 (fr) * 2021-06-01 2022-12-08 Nanovation Therapeutics Inc. Administration de vecteur d'adn à l'aide de nanoparticules lipidiques

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ALISON ET AL., BIOCHIM BIOPHYS ACTA, vol. 1468, no. 1-2, 29 September 2000 (2000-09-29), pages 127 - 38
ARMSTRONGANCHORDOQUY, J PHARM SCI., vol. 93, no. 11, November 2004 (2004-11-01), pages 2698 - 709
BATZER ET AL., NUCLEIC ACID RES., vol. 19, 1991, pages 5081
KULKARNI JAYESH A ET AL: "Design of lipid nanoparticles forin vitroandin vivodelivery of plasmid DNA", NANOMEDICINE: NANOTECHNOLOGY, BIOLOGY, AND MEDICINE, ELSEVIER, AMSTERDAM, NL, vol. 13, no. 4, 28 December 2016 (2016-12-28), pages 1377 - 1387, XP085043397, ISSN: 1549-9634, DOI: 10.1016/J.NANO.2016.12.014 *
OHTSUKA ET AL., J. BIOL. CHEM., vol. 260, 1985, pages 2605 - 2608
ROSSOLINI ET AL., MOL. CELL. PROBES, vol. 8, 1994, pages 91 - 98

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