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WO2018119514A1 - Compositions pour la transfection de types de cellules résistantes - Google Patents

Compositions pour la transfection de types de cellules résistantes Download PDF

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Publication number
WO2018119514A1
WO2018119514A1 PCT/CA2017/051543 CA2017051543W WO2018119514A1 WO 2018119514 A1 WO2018119514 A1 WO 2018119514A1 CA 2017051543 W CA2017051543 W CA 2017051543W WO 2018119514 A1 WO2018119514 A1 WO 2018119514A1
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lipid
composition
nucleic acid
mol
cell
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PCT/CA2017/051543
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English (en)
Inventor
Anitha THOMAS
Rebecca Anne Grace DE SOUZA
Eric Ouellet
Grace Tharmini THARMARAJAH
Jagbir Singh
Shyam Madhusudan GARG
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Precision Nanosystems Inc.
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Priority to US16/349,274 priority Critical patent/US11572575B2/en
Publication of WO2018119514A1 publication Critical patent/WO2018119514A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/35—Polyols, e.g. glycerin, inositol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]

Definitions

  • the field of the invention is the transfer of active nucleic acids into cells.
  • Nucleic acids in the form of polynucleotides or oligonucleotides can be used to focus treatment on a particular genetic target, either by interfering with its expression, or by restoring or augmenting its expression, or by editing the gene.
  • nucleic acids are relatively large, negatively charged, hydrophilic compounds which are not capable of passively diffusing across the cell membrane and are also vulnerable to nucleases.
  • LNP lipid nanoparticles
  • ionisable cationic lipids also known as "cationic lipids”
  • PEGylated lipids for the efficient encapsulation and delivery of nucleic acids to cells
  • LNPs have been engineered to obtain different pharmacokinetics, different bio- distribution in tissues, biodegradability, or altered toxicity, to favor the therapeutic activity of the nucleic acid.
  • Triolein has been tested in l-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine micelle size-ranging experiments.
  • CNS tissue is notoriously difficult to transfect (O'Mahony, Godinho et al. 2013).
  • the cells are sensitive to their conditions and are hard to maintain in vitro. A more effective method for effectively delivering nucleic acids into neurons is still required.
  • a composition for transfecting nucleic acid into live cells including: 30-50 mol % of a cationic lipid or pharmaceutically acceptable salt thereof; 10-50 mol % of a structural lipid; 5 to 40% mol % of a triglyceride; and 0.1 to about 10 mol % of a stabilizing agent.
  • the cationic lipid is an amino lipid or a pharmaceutically acceptable salt thereof.
  • the cationic lipid is l,17-bis(2-octylcyclopropyl)heptadecan-9- yl-4-(dimethylamino).
  • the structural lipid is selected from diacylphosphatidylcholines, diacylphosphatidylethanola mines, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides.
  • the triglyceride is selected from the group consisting of triolein, tristearin, trilaurin, trilinoein, trilinolenin, trimyristin, tripalmitin, tricaprylin, and oleoyldipalmitin.
  • the stabilizing agent is selected from PEG-lipid conjugates, polyoxyethylene alkyl ethers, diblock polyoxyethylene ether co-polymers, triblock polyoxyethylene alkyl ethers co-polymers, and amphiphilic branched polymers.
  • the PEG-lipid is selected from the group consisting of PEG-ceramide, PEG- DMG, PEG-PC, PEG-PE, and PEG-DMA.
  • the stabilizing agent is selected from polyoxyethylene (20) oleyl ether, polyoxyethylene (23) lauryl ether, polyoxyethylene (40) stearate ("Myrj52"), poly(propylene glycol)ll-block-poly(ethylene glycol)16-block-poly(propylene glycol)ll, poly(propylene glycol)12-block-poly(ethylene glycol)28-block-poly(propylene glycol)12.
  • the composition further including a sterol, for example, cholesterol.
  • the cationic lipid is about 40 mol% of the composition.
  • the triglyceride is from about 1 to about
  • the cationic lipid is l,17-bis(2- octylcyclopropyl)heptadecan-9-yl-4-(dimethylamino) butanoate or a pharmaceutically acceptable salt thereof.
  • the structural lipid makes up
  • the triglyceride is triolein.
  • the stabilizing agent is 1 to 5 mol % polyoxyethylene (40) stearate (a.k.a. "Myrj52").
  • the stabilizing lipid includes about 2.5 mole percent of polyoxyethylene (40) stearate.
  • the structural lipid is DOPE. In embodiments of the invention, the structural lipid is DSPE.
  • the composition further includes a nucleic acid.
  • the nucleic acid is DNA. In other embodiments, the nucleic acid is an
  • the nucleic acid is a locked nucleic acid.
  • the nucleic acid is a nucleic acid analog.
  • the nucleic acid is a plasmid.
  • the nucleic acid is circular.
  • the nucleic acid is linear.
  • the composition exists in the form of nanoparticles having a diameter of from about 15nm to about 300nm.
  • a method for introducing a nucleic acid into a cell is provided, which method maintains activity of the nucleic acid and viability of the cell.
  • the method include contacting the cell with the embodiment compositions.
  • a method for modulating the expression of a target polynucleotide or polypeptide in a cell while maintaining cell viability includes contacting the cell with the composition embodiments of the invention.
  • the cell is a neuron.
  • the cell is a mammalian cell.
  • the transfecting occurs in vitro. In embodiments, the transfecting occurs in vivo.
  • Figure 1 is a bar graph of in vitro neuron viability demonstrated by PrestoBlue assay under seven conditions at 48h: no treatment, treatment with Lipid Mix G GFP mRNA LNP, treatment with Lipid Mix H GFP mRNA LNP and treatment with commercial reagent MessengerMaxTM LipofectamineTM (2.5 ug/mL or 5 ug/mL of LNP, 1 ug/mL or 2.5 ug/mL of Messenger Max) + 5 ug/mL ApoE;
  • Figure 2 is a graphical representation of percentage knock down of HPRT mediated by siRNA delivery to neurons using negative control siRNA (hereinafter "NC- 1"), HPRT siRNA Lipid A LNP; HPRT siRNA Lipid Mix G LNP and HPRT siRNA Lipid Mix H LNP. HPRT expression is shown as a percent of untreated control (UT) and normalized to Beta actin (ActB);
  • Figure 3 is a black and white, image reversed photograph of neurons that have been exposed to Lipid Mix A LNP containing GFP mRNA (left panel), and Lipid Mix G formulated nanoparticles containing GFP mRNA (right panel).
  • MAP2 antibody staining shows the outline of the neurons
  • DAPI stain shows the nuclei
  • GFP expression in the neurons is shown as darkest staining.
  • Merged images showing DAPI, MAP2, and GFP staining establish GFP expression in the live neurons treated with Lipid Mix G LNP;
  • Figure 4 is a graphical representation of the results obtained by flow cytometry analysis of neurons treated with Lipid Mix A and G encapsulating GFP mRNA.
  • the first figure is a bar graph corresponding to the percentage of neurons positive for GFP
  • the second figure is a bar graph showing the mean fluorescent intensity of GFP expression in neurons treated with Lipid Mix A and Lipid Mix G nanoparticles (2.5 ug/mL of LNP + 5 ug/mL ApoE);
  • Figure 5 is a graphical representation of the results obtained by flow cytometry analysis of neurons treated with Lipid Mix A and Lipid Mix H nanoparticles encapsulating GFP mRNA.
  • the upper bar graph corresponds to the percentage of neurons that are positive for GFP
  • the lower bar graph shows the mean fluorescent intensity of GFP expression in neurons treated with Lipid Mix A and Lipid Mix H nanoparticles (2.5 ug/mL of LNP + 5 ug/mL
  • Figure 6 is a graphical plot of GFP levels in pg/mL, measured by ELISA performed on
  • the four bars represent the amount of GFP detected in untreated neurons, in neurons treated with Lipid Mix A, Lipid Mix G and Lipid M ix H mRNA LNP respectively from left to right;
  • Figure 7 is a graphical representation of flow cytometry of GFP-expressing plasmid
  • the mean fluorescent intensity of MFI indicates the amount of GFP expression mediated by different nanoparticle formulations.
  • concentrations of treatment nanoparticles was lug/ml of LNP with 5ug/ml ApoE;
  • Figure 8 is a graphical rendition of flow cytometry viability data of DIV7 neurons exposed to the various plasmid DNA nanoparticles, measured 48h post exposure. Data is shown for plasmid containing lipid nanoparticles untreated control, Lipid Mix G, and Lipid Mix H at 1 ug/mL LNP and 5 ug/mL ApoE; and
  • Figure 9 is a graphical representation of GFP expression in vivo in pg/mL, in spleen from the mRNA treated C57BL/6 mice at a dose of lmg/kg (iv) using particles of Lipid Mix D and Lipid Mix H; GFP expression was quantified using GFP ELISA kit.
  • the present invention provides compositions, lipid nanoparticles containing a therapeutic agent, methods for making the lipid nanoparticles containing a therapeutic agent, methods for targeting specific cell types, and methods for delivering a therapeutic agent using the lipid nanoparticles.
  • a lipid mix composition that includes one or more cationic lipid(s), one or more structural lipid(s), and one or more stabilizing agent(s).
  • the lipid mix compositions of the invention are provided for mixing with nucleic acid therapeutics for delivery to target cells or tissues, or for treatment of mammals in need of such delivery for treatment of insufficiency or disease.
  • the lipid mix further includes one or more triglyceride(s).
  • the lipid mix compositions according the invention are provided for formulating nucleic acid therapeutics for the treatment of diseases of the CNS.
  • Neuron in this application mean mature neurons, neural progenitor cells, and are characterized by electrical excitability and the presence of membrane junctions that transmit signals to other cells (synapses).
  • Lipid refers to a group of organic compounds that are based on fatty acids, and are characterized by being insoluble in water but soluble in many organic solvents.
  • the invention provides lipid nanoparticles manufactured from the compositions described above.
  • the lipid nanoparticles contain a therapeutic agent in some embodiments.
  • the lipid nanoparticles include one or more cationic lipids, one or more structural lipid(s), one or more stabilizing agent(s), and one or more nucleic acids.
  • the lipid nanoparticles include a cationic lipid.
  • cationic lipid refers to a lipid that is cationic or becomes ionizable (protonated) as the pH is lowered below the pK of the cationic group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids (e.g., oligonucleotides).
  • cationic lipid includes zwitterionic lipids that assume a positive charge on pH decrease, and any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH.
  • lipids include, but are not limited to N,N- dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N— (N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), C12-200 and N-(l,2- dimyristyloxyprop-3-
  • DODAC N,
  • the cationic lipid is an amino lipid.
  • Suitable amino lipids useful in the invention include those described in WO 2009/096558, incorporated herein by reference in its entirety.
  • Representative amino lipids include l,17-bis(2- octylcyclopropyl)heptadecan-9-yl 4-(dimethylamino)butanoate hydrochloride, 1,2- dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), l,2-dilinoleyoxy-3- morpholinopropane (DLin-MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP),
  • Ri and R 2 are either the same or different and independently optionally substituted Ci 0 -C 2 4 alkyl, optionally substituted Ci 0 -C 2 4 alkenyl, optionally substituted C 10 -C24 alkynyl, or optionally substituted Ci 0 -C 2 4 acyl;
  • R 3 and R 4 are either the same or different and independently optionally substituted Ci-C 6 alkyl, optionally substituted C 2 -C 6 alkenyl, or optionally substituted C 2 -C 6 alkynyl or R 3 and R 4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen;
  • R 5 is either absent or present and when present is hydrogen or Ci-C 6 alkyl
  • n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0;
  • q is 0, 1, 2, 3, or 4; and Y and Z are either the same or different and independently 0, S, or NH .
  • Ri and R 2 are each linoleyl, and the amino lipid is a dilinoleyl amino lipid.
  • the amino lipid is a d ilinoleyl amino lipid.
  • the cationic lipid is l,17-bis(2- octylcyclopropyl)heptadecan-9-yl 4-(dimethylamino)butanoate hyd rochloride. This compound is disclosed in United States Published Application 2013323269.
  • the cationic lipid-like material is C12-200 as described by Kaufmann and his colleagues (Kaufmann k 2015)
  • the cationic lipid is present in embodiments of the composition and lipid particle of the invention comprise an amount from about 30 to about 60 mole percent ( "MOL%", or the percentage of the total moles that is of a particular component), preferably from 30 to 50MOL%. In preferred embod iments, the cationic lipid is present in 40MOL%.
  • Structural lipids The composition and lipid nanoparticles of the invention include one or more structural lipids. Suitable structural lipids support the formation of particles during manufacture. Structural lipids refer to any one of a number of lipid species that exist in either in an anionic, uncharged or neutral zwitterionic form at physiological pH. Representative structural lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, diacylphosphatidylglycerols, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides.
  • Exemplary structural lipids include zwitterionic lipids, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), pa I mitoyloleoyl phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearioy
  • the structural lipid is l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE).
  • the structural lipid may be any lipid that is negatively charged at physiological pH.
  • lipids include phosphatidylglycerol such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyolphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, other anionic modifying groups joined to neutral lipids.
  • DOPG dioleoylphosphatidylglycerol
  • DPPG dipalmitoylphosphatidylglycerol
  • POPG palmitoyloleyolphosphatidylglycerol
  • cardiolipin phosphatidylinositol
  • diacylphosphatidylserine diacylphosphatidic acid
  • Stabilizing Agents are included in compositions and lipid nucleic acid embodiments to ensure integrity of the mixture.
  • Stabilizing agents are polyethylene glycol-lipids.
  • Suitable polyethylene glycol-lipids include PEG- modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerCi 4 or PEG-CerC 2 o), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols.
  • Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG.
  • the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol) 2 ooo)carbamyl]-l,2- dimyristyloxlpropyl-3-amine (PEG-c-DMA).
  • the polyethylene glycol-lipid is PEG-c-DOMG. In another embodiment, the polyethylene glycol-lipid is PEG-DMG (1,2-Dimyristoyl-sn-glycerol, methoxypolyethylene glycol). In some embodiments, the polyethylene glycol lipid content is from 0 to 10% of the Lipid Mix.
  • the stabilizing agent is polyoxyethylene alkyl ether, diblock polyoxyethylene ether co-polymer, triblock polyoxyethylene alkyl ethers co-polymer, or amphiphilic branched polymers.
  • the stabilizing agent is polyoxyethylene (20) oleyl ether, polyoxyethylene (23) lauryl ether, polyoxyethylene (40) stearate, poly(propyleneglycol) 11-block-poly (ethyleneglycol) 16-block- poly(propylene glycol)ll, poly(propylene glycol)12-block-poly( ethylene glycol)28-block- poly(propylene glycol)12.
  • Sterols are included in some embodiments of the compositions, and lipid nanoparticles made therefrom include sterols, such as cholesterol.
  • Other suitable structural lipids include glycolipids (e.g., monosialoganglioside GMi).
  • the structural lipid is present in the lipid particle in an amount from about 20 to about 40 MOL.%. In one embodiment, the structural lipid is present in the lipid particle in an amount from about 20 to about 30 MOL.%. In one embodiment, the structural lipid is present in the lipid particle in about 20 MOL%, or in about 30
  • the cationic lipid was l,17-bis(2-octylcyclopropyl)heptadecan-9-yl 4-(dimethylamino)butanoate hydrochloride and the structural lipid (DOPE, for example) was 20, 30 or 40 MOL%.
  • DOPE structural lipid
  • the addition of 10 to 20 mole percent of triolein to the lipid mix resulted in increased target cell viability when added to compositions and lipid nucleic acid particles of the invention.
  • the addition of this triglyceride also surprisingly enhanced mRNA expression in target cells.
  • Triglycerides are esters of fatty acids with glycerol. Tristearin, trilaurin, trilinoein, trilinolenin, trimyristin, tripalmitin, tricaprylin, oleoyldipalmitin are examples. All three fatty acids can be the same or different. Triglyceride can be symmetric or asymmetric. SSS, SUS, and USU- triglycerides are considered symmetric triglycerides, where S represents a saturated fatty acid and U represents an unsaturated fatty acid. If the sn-1 and sn-3 position contain different fatty acids, then the central carbon atom is a chiral carbon, and is asymmetric.
  • a fatty acid is a carboxylic acid with a long aliphatic chain consisting of 4-28 even number of carbons, comprising short, medium or long chain saturated or unsaturated triglyceride, given embodiment may contain more importantly 12 -22 carbon atoms.
  • Examples of common fatty acids include, but are not limited to, linoleic acid, linolenic acid, oleic acid, palmitic acid, lauric acid, stearic acid, behenic acid, arachidonic acid, etc.
  • the fatty acid can be unsaturated or saturated.
  • Triglycerides can be naturally derived or synthesized.
  • Example of natural origin fatty acids include omega 3 fatty acid, omega 6 fatty acid, etc.
  • omega3 fatty acids examples include alpha- linolenic acid , docasahexaenoic acid.
  • omega6 fatty acids examples include linoleic acid, and gamma linolenic acid.
  • Triglycerides can also be part of compounds classified under the following trade names: Captex ® , Sterotex ® , and others.
  • the triglyceride is triolein (TO), a symmetrical triglyceride derived from glycerol and three units of the unsaturated fatty acid oleic acid.
  • TO triolein
  • the lUPAc name for triolein is 2,3-bis[[(Z)-octadec-9-enoyl]oxy]propyl (Z)-octadec- 9-enoate, and synonyms include glycerol trioleate, glycerol, trioleyl, trielaidin, trioleoylglycerol, and trioleyl glycerol.
  • triolien is replaced by another symmetrical triglyceride such as one or a mixture of tristearin, trilaurin, trilinoein, trilinolenin, trimyristin, tripalmitin, tricaprylin, or oleoyldipalmitin.
  • another symmetrical triglyceride such as one or a mixture of tristearin, trilaurin, trilinoein, trilinolenin, trimyristin, tripalmitin, tricaprylin, or oleoyldipalmitin.
  • a mix of triglycerides are included in the lipid mix composition.
  • nucleic Acids The lipid mix compositions and lipid nanoparticles of the present invention are useful for the systemic or local delivery of nucleic acid therapeutics.
  • the nucleic acid therapeutic NAT is incorporated into the lipid particle during its formation.
  • nucleic acid therapeutic is meant to include any oligonucleotide or polynucleotide whose delivery into a cell causes a desirable effect. Fragments containing up to 50 nucleotides are generally termed oligonucleotides, and longer fragments are called polynucleotides. In particular embodiments, oligonucleotides of the present invention are 20-50 nucleotides in length. In other embodiments of the invention, oligonucleotides are 996 to 4500 nucleotides in length, as in the case of messenger RNA or plasmids.
  • polynucleotide and “oligonucleotide” refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar
  • polynucleotide and “oligonucleotide” also include polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as enhanced cellular uptake and increased stability in the presence of nucleases. Oligonucleotides are classified as deoxyribooligonucleotides or ribooligonucleotides. A deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose joined covalently to phosphate at the 5' and 3' carbons of this sugar to form an alternating, unbranched polymer.
  • a ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose.
  • the nucleic acid that is present in a lipid particle according to this invention includes single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids.
  • double-stranded DNA include structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA.
  • double-stranded RNA include siRNA and other RNA interference reagents.
  • Single-stranded nucleic acids include antisense oligonucleotides, ribozymes, microRNA, mRNA, and triplex-forming oligonucleotides.
  • the polynucleic acid is an antisense oligonucleotide.
  • the nucleic acid is a ribozyme, a non-coding nuclear or nucleolar RNA as explained in HUGO http://www.genenames.org/cgi-bin/genefamilies, imiRNA, rRNA, tRNA, siRNA, saRNA, snRNA, snoRNA, IncRNA, piRNA, tsRNA, srRNA, crRNA, tracrRNA, sgRNA, shRNA, ncRNA, imiRNA, mRNA, pre-condensed DNA, pDNA, an aptamer, or a combination thereof.
  • the nucleic acid therapeutic is a plasmid or circular nucleic acid construct.
  • the NAT is a mRNA.
  • the NAT is a siRNA.
  • the NAT is a imiRNA.
  • the NAT is a tracrRNA.
  • the NAT is a sgRNA.
  • nucleic acids also refers to ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar-backbone oligonucleotides, other nucleotides, nucleotide analogs, and combinations thereof, and can be single stranded, double stranded, or contain portions of both double stranded and single stranded sequence, as appropriate.
  • the nucleic acid exists in a circular plasmid structure.
  • polynucleotide and “oligonucleotide” are used interchangeably and mean single-stranded and double-stranded polymers of nucleotide monomers, including 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bond linkages, e.g., 3'-5' and 2'-5', inverted linkages, e.g., 3'-3' and 5'-5', branched structures, or internucleotide analogs.
  • DNA 2'-deoxyribonucleotides
  • RNA ribonucleotides linked by internucleotide phosphodiester bond linkages, e.g., 3'-5' and 2'-5', inverted linkages, e.g., 3'-3' and 5'-5', branched structures, or internucleotide analogs.
  • Polynucleotides have associated counter ions, such as H+, NH4+, trialkylammonium, Mg2+, Na+, and the like.
  • a polynucleotide may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof.
  • Preferred nucleic acids are DNA and RNA.
  • nucleic acids may also refer to "peptide nucleic acid” or "PNA” means any oligomer or polymer segment (e.g., block oligomer) comprising two or more PNA subunits (residues), but not nucleic acid subunits (or analogs thereof), including, but not limited to, any of the oligomer or polymer segments referred to or claimed as peptide nucleic acids in U.S. Pat. Nos. 5,539,082; 5,527,675; 5,623,049; 5,714,331; 5,718,262;
  • peptide nucleic acid also applies to any oligomer or polymer segment comprising two or more subunits of those nucleic acid mimics such as those described in Peptide-Based Nucleic Acid Mimics (PENAMS) of Shah et al. as disclosed in Peptide-Based Nucleic Acid Mimics (PENAMS) of Shah et al. as disclosed in Peptide-Based Nucleic Acid Mimics (PENAMS) of Shah et al. as disclosed in
  • the lipid nanoparticles (LNP) can also be characterized by electron microscopy.
  • the LNP of the invention have a substantially solid core with an electron dense core when viewed by electron microscopy.
  • Electron density is calculated as the absolute value of the difference in image intensity of the region of interest from the background intensity in a region containing no nanoparticle.
  • the lipid nanoparticles of the invention have a diameter (mean particle diameter) from about 15 to about 300 nm. In some embodiments, the mean particle diameter is greater than 300 nm. In some Embodiments, the lipid particle has a diameter of about 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, or 50 nm or less. I n one embodiment, the lipid particle has a diameter from about 50 to about 150 nm. These smaller particles generally exhibit increased circulatory lifetime in vivo compared to large particles. I n one embod iment, the lipid particle has a diameter from about 15 to about 50 nm.
  • the lipid nanoparticles of the invention are substantially homogeneous in their size d istribution.
  • the lipid nanoparticles of the invention have a mean particle diameter standard deviation of from about 65 to about 25%.
  • the lipid nanoparticles of the invention have a mean particle d iameter standard deviation of about 60, 50, 40, 35, or 30%.
  • the lipid nucleic acid particles of the invention have a PDI of about 0.01 to 0.3.
  • the lipid nanoparticles according to embodiments of the invention have nearly 100% of the nucleic acid used in the formation process is encapsulated in the nanoparticles.
  • the lipid nanoparticles have about 90 to about 95% of the nucleic acid used in the formation process encapsulated in the lipid nanoparticles (LNP).
  • the invention provides a method for making LNP containing a therapeutic agent.
  • LNP systems containing genetic drugs A variety of manual methods have been developed to formulate LNP systems containing genetic drugs. These methods include mixing preformed LN P with nucleic acid therapeutic (NAT) in the presence of ethanol, or mixing lipid dissolved in ethanol with an aqueous media containing NAT, and result in LNP with NAT encapsulation efficiencies of 65-95%.
  • the mixing can be done in bulk, or using pipette mixing, or in a "T Tube" with two streams of reagents meeting the middle and running down a pipe together. Both of these methods rely on the presence of cationic lipid to achieve encapsulation of NAT, and a stabilizing agent to inhibit aggregation and the formation of large structures.
  • the properties of the LNP systems produced are sensitive to a variety of formulation parameters such as ionic strength, lipid and ethanol concentration, pH, NAT concentration and mixing rates.
  • formulation parameters such as ionic strength, lipid and ethanol concentration, pH, NAT concentration and mixing rates.
  • parameters such as the relative lipid and NAT concentrations at the time of mixing, as well as the mixing rates, are difficult to control using manual formulation procedures, resulting in variability in the characteristics of LNP produced, both within and between preparations.
  • Microfluidic mixing devices such as the NanoAssemblrTM series by Precision NanoSystems Inc. (Vancouver, Canada) enable controlled and rapid mixing of fluids on a nanoliter scale. Temperature, residence time, and solute concentration are controlled during the process of rapid microfluidic mixing applied in the synthesis of inorganic lipid particles, and can outperform manual systems in large scale production of nanoparticles. Microfluidic two-phase droplet techniques have been applied to produce monodisperse polymeric microparticles for drug delivery or to produce large vesicles for the encapsulation of cells, proteins, or other biomolecules.
  • hydrodynamic flow focusing a common microfluidic technique to provide rapid mixing of reagents, to create monodisperse liposomes of controlled size has been demonstrated. This technique has also proven useful in the production of polymeric nanoparticles where smaller, more monodisperse particles were obtained, with higher encapsulation of small molecules as compared to bulk production methods.
  • U.S. Application Pub. No. 20160022580 by Ramsay et al. describes more advanced methods of using small volume mixing technology and products to formulate different materials.
  • U.S. Application Pub. No. 2016235688 by Walsh, et al. discloses microfluidic mixers with different paths and wells to elements to be mixed.
  • PCT Publication WO2017117647 discloses microfluidic mixers with disposable sterile paths.
  • a suitable microfluidic mixing device includes one or more microchannels (i.e., a channel having its greatest dimension less than 1 millimeter).
  • the microchannel has a diameter from about 20 to about 300 ⁇ .
  • at least one region of the microchannel has a principal flow direction and one or more surfaces having at least one groove or protrusion defined therein, the groove or protrusion having an orientation that forms an angle with the principal direction (e.g., a staggered herringbone mixer), as described in U.S. Application Pub. No. 2004/0262223.
  • at least one region of the microchannel has bas-relief structures.
  • the lipid nanoparticles of the present invention may be used to deliver a therapeutic agent to a cell, in vitro or in vivo.
  • the therapeutic agent is a nucleic acid, which is delivered to a cell using nucleic acid-lipid nanoparticles of the present invention.
  • the methods and compositions may be readily adapted for the delivery of any suitable therapeutic agent for the treatment of any disease or disorder that would benefit from such treatment.
  • the present invention provides methods for introducing a nucleic acid into a cell.
  • Preferred nucleic acids for introduction into cells are siRNA, mRNA, immune-stimulating oligonucleotides, plasmids, antisense and ribozymes. These methods may be carried out by contacting the particles or compositions of the present invention with the cells for a period of time sufficient for intracellular delivery to occur.
  • Typical applications include using well known procedures to provide intracellular delivery of siRNA to knock down or silence specific cellular targets.
  • Alternatively applications include delivery of DNA or mRNA sequences that code for therapeutically useful polypeptides. In this manner, therapy is provided for genetic diseases by supplying deficient or absent gene products.
  • Methods of the present invention may be practiced in vitro, ex vivo, or in vivo.
  • the compositions of the present invention can also be used for delivery of nucleic acids to cells in vivo, using methods which are known to those of skill in the art.
  • siRNA, mRNA and plasmid nucleic acid therapeutics by a lipid particle of the invention is described below.
  • the pharmaceutical compositions are preferably administered parenterally (e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intradermaly, intratrachealy, intraosseous or intramuscularly).
  • parenterally e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intradermaly, intratrachealy, intraosseous or intramuscularly.
  • the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection.
  • Other routes of administration include topical
  • the present invention provides a method of modulating the expression of a target polynucleotide or polypeptide. These methods generally comprise contacting a cell with a lipid particle of the present invention that is associated with a nucleic acid capable of modulating the expression of a target polynucleotide or polypeptide.
  • modulating refers to altering the expression of a target polynucleotide or polypeptide. Modulating can mean increasing or enhancing, or it can mean decreasing or reducing.
  • the present invention provides a method of treating a disease or disorder characterized by overexpression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition of the present invention, wherein the therapeutic agent is selected from an siRNA, a microRNA, an antisense oligonucleotide, and a plasmid capable of expressing an siRNA, a microRNA, or an antisense oligonucleotide, and wherein the siRNA, microRNA, or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide that encodes the polypeptide, or a complement thereof.
  • the therapeutic agent is selected from an siRNA, a microRNA, an antisense oligonucleotide, and a plasmid capable of expressing an siRNA, a microRNA, or an antisense oligonucleotide
  • the siRNA, microRNA, or antisense RNA comprises a polynucleotide that specifically
  • the present invention provides a method of treating a disease or disorder characterized by underexpression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition of the present invention, wherein the therapeutic agent is selected from an mRNA, a self-replicating DNA, or a plasmid, comprises a nucleic acid therapeutic that specifically encodes or expresses the under-expressed polypeptide, or a complement thereof.
  • Exemplary mRNA encodes the protein or enzyme selected from human growth hormone, erythropoietin, a 1 -antitrypsin, acid alpha glucosidase, arylsulfatase A, carboxypeptidase N, a-galactosidase A, alpha-L-iduronidase, iduronate-2- sulfatase, iduronate sulfatase, N-acetylglucosamine- 1 -phosphate transferase, N- acetylglucosaminidase, alpha-glucosaminide acetyltransferase, N- acetylglucosamine 6- sulfatase, N-acetylgalactosamine-4-sulfatase, beta- glucosidase, galactose-6-sulfate sulfatase, beta-galactosidase,
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a lipid particle of the invention and a pharmaceutically acceptable carrier or diluent.
  • pharmaceutically acceptable carriers or diluents include solutions for intravenous injection (e.g., saline or dextrose).
  • the composition can take the form of a cream, ointment, gel, suspension, or emulsion.
  • LNP lipid nucleic acid particle
  • Formulation of LNPs was performed by rapidly mixing a lipid-ethanol solution with an aqueous buffer inside a microfluidic mixer designed to induce chaotic advection and provide a controlled mixing environment at intermediate Reynolds number (24 ⁇ Re ⁇ 1000).
  • the microfluidic channel has herringbone features or configured in a manner as shown in PCT Pub. No. WO/2017/117647 by Wild, Leaver and Taylor .
  • Particle sizes and "polydispersity index" (PDI) of the LNP were measured by dynamic light scattering (DLS).
  • PDI indicates the width of the particle distribution. This is a parameter calculated from a cumulative analysis of the (DLS)-measured intensity autocorrelation function assuming a single particle size mode and a single exponential fit to the autocorrelation function. From a biophysical point of view, a PDI below 0.1 indicates that the sample is monodisperse (As a reference, PDI of the NIST standards are below 0.05.) .
  • the following embodiments are provided for the purpose of illustrating, not limiting, the claimed invention.
  • a stabilizing agent is present in the particle in an amount from about 0.1 to about 20 MOL%.
  • the stabilizing agent is a surfactant.
  • the stabilizing agent is a PEG Lipid.
  • the stabilizing agent is present in the particle in an amount from about 0.5 to about 10MOL%.
  • the stabilizing agent is present in the lipid nanoparticle at about 2.5MOL%.
  • the stabilizing agent is polyoxyethylene (40) stearate.
  • the stabilizing agent is polyoxyethylene (20) oleyl ether. "Myrj52TM" is a tradename for polyoxyethylene (40) stearate. It is sold by Sigma-Aldrich Canada Co .
  • the stabilizing agent is a polyethylene glycol-lipid.
  • Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG- modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols.
  • Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c- DMA, and PEG-s-DMG. Many of these are for sale from NOF America Corporation, White Plains, NY under brand names such as SUNBRIGHT ® GM-020 (DMG-PEG)
  • stabilizing agent can be a non-ionic surfactant.
  • the Stabilizing agent is a PEG-lipid is present at concentrations of 0 to 10 MOL.%.
  • stabilizing agent can be vitamin E TPGS.
  • the repeating PEG moiety has an average molecular weight of 1000 - 2000.
  • molecular weight average of the PEG segment is 1700 - 2000 with n value (number of PEG repeating units) between 39 - 47.
  • the nucleic acid is a plasmid composed of double stranded deoxyribonucleic acid.
  • a plasmid is a genetic structure that resides in a cell's cytoplasm (as opposed to the nucleic where the traditional cellular genetics reside) cell that can replicate independently of the chromosomes, typically a small circular DNA strand. This is not a normal mammalian genetic construct, but is used as a therapeutic option for replacing or restoring faulty genetic function in a cell. Plasmids can also be used to create novel cellular or animal models for medical research.
  • An engineered plasmid will have, in addition to a replication origin (or not, depending on the intended use), restriction enzyme recognition sites, which allow breaking the circle to introduce new genetic material, and a selective marker such as an antibiotic resistance gene.
  • a plasmid may be from 2,000 to about 1 milion base pairs (bp). The larger the plasmid, the more susceptible it is to shearing forces, which increases the need for encapsulation.
  • the term "about” is defined as meaning 10% plus or minus the recited number. It is used to signify that the desired target concentration might be, for example, 40 mol %, but that through mixing inconsistencies, the actual percentage might differ by +/- 5 mol %.
  • mole % or mol % relates to the number of atoms or molecules of each component, rather than just the mass. Specifically, 1 mole represents 6.022 x 10 23 atoms or molecules of substance. Moles are calculated by dividing the mass of the component by the component's atomic or molecular weight (which is determined by adding all of the atomic masses for the atoms in a chemical formula as found on the periodic table of the elements). The mole fraction is determined by dividing the moles of one component in a mixture by the total number of moles of all substances in the mixture. The mole fractions for all substances in a mixture will add up to 100 percent.
  • nucleic acid is defined as a substance intended to have a direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to have direct effect in restoring, correcting or modifying physiological functions, or to act as a research reagent.
  • the nucleic acid is an oligonucleotide.
  • the therapeutic agent is a nucleic acid therapeutic, such as an RNA polynucleotide.
  • the therapeutic agent is double stranded circular DNA (plasmid).
  • the term "research reagent” is defined by the fact that it has a direct influence on the biological effect of cells, tissues or organs.
  • Research reagents include but are not limited polynucleotides, proteins, peptides, polysaccharides, inorganic ions and radionuclides.
  • nucleic acid research reagents include but are not limited to antisense oligonucleotides, ribozymes, microRNA, mRNA, ribozyme, tRNA, tracrRNA, sgRNA, snRNA, siRNA, shRNA, ncRNA, miRNA, mRNA, pre-condensed DNA, pDNA or an aptamer.
  • Nucleic acid Research Reagents are used to silence genes (with for example siRNA), express genes (with for example mRNA), edit genomes (with for example CRISPR/Cas9).
  • the word "comprising” is used in a non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It will be understood that in embodiments which comprise or may comprise a specified feature or variable or parameter, alternative embodiments may consist, or consist essentially of such features, or variables or parameters. A reference to an element by the indefinite article “a” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements.
  • transfecting reagent means a composition that enhances the transfer of nucleic acid into cells. It typically includes a cationic lipid to associate with nucleic acid, and structural lipids.
  • LIPOFECTINTM and LIPOFECTAMINETM are legacy transfecting reagents incorporating cationic lipids like DOTMA.
  • MESSENGER MAXTM LIPOFECTAMINETM is a contemporary transfecting reagent sold by ThermoFisher.
  • Lipofectamine ® MessengerMAXTM reagent is designed to transfect a higher amount of mRNA into neurons and difficult-to-transfect primary cells.
  • the recitation of numerical ranges by endpoints includes all numbers subsumed within that range including all whole numbers, all integers and all fractional intermediates (e.g., 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.80, 4, and 5 etc.).
  • the singular forms an "an”, and “the” include plural referents unless the content clearly dictates otherwise.
  • reference to a composition containing "a compound” includes a mixture of two or more compounds.
  • Ammonium acetate, sodium acetate and sodium chloride were obtained from Fisher Scientific (Fair Lawn, NJ). Cholesterol, stabilizer lipid "Myrj52TM” or PEG (40) stearate, and triolein were purchased from Sigma-Aldrich (Oakville, Ontario). DiD is a lipid label used for visualizing lipid components. It is DilC 18 (5) stain (l,l'-Dioctadecyl-3,3,3',3'- Tetramethylindodicarbocyanine Perchlorate and available from Invitrogen
  • the mRNA used was Enhanced Green Fluorescent Protein messenger RNA (EGFP mRNA) (5-methylcytidine, pseudouridine, Length: 996 nucleotides (SEQ ID No. 1) (from Trilink Biotechnologies, San Diego, CA), at 1.0 mg/mL in 10 mM Tris-HCI, pH 7.5.
  • EGFP mRNA Enhanced Green Fluorescent Protein messenger RNA
  • SEQ ID No. 1 from Trilink Biotechnologies, San Diego, CA
  • the unknown bases in SEQ ID NO. 1 are a portion of alpha globin which is a vendor trade secret.
  • RNase A was obtained from Applied Biosystems/Ambion (Austin, TX).
  • the siRNA used was Hypoxanthine phosphoribosyltransferase 1 siRNA rG rCrCrArG rArCrU rU rU rG rU rU rG rG rArU rU rG rArU rU rG rArArATT
  • DsiRNA dicer-substrate siRNA
  • HPRT SEQ ID NO 2, sense, SEQ ID NO 3, antisense
  • control siRNA DSiNC-1 a nonsilencing, negative control DsiRNA that does not recognize any sequences in human, mouse, or rat transcriptomes (SEQ ID NO 4, sense, SEQ ID NO 5, antisense).
  • DsiRNA and DsiNCl were purchased from Integrated DNA Technologies, Inc., Coralville, Iowa).
  • Structural lipid DOPE structural lipid DSPC
  • ionisable lipid l,17-bis(2- octylcyclopropyl)heptadecan-9-yl 4-(dimethylamino) butanoate were purchased from Avanti Polar Lipids (Alabaster, AL, USA),
  • Oligonucleotide or polynucleotide (siRNA, mRNA or plasmid, hereinafter referred to as "nucleic acid”) solution was prepared in 25 mM-100 mM acetate buffer at pH 4.0. Depending on the desired oligonucleotide-to-lipid ratio and formulation concentration, solutions were prepared at a target concentration of 2.3 mg/ml to 4 mg/ml total lipid.
  • DiD label was prepared in ethanol and mixed with the oligonucleotide or polynucleotide to achieve an ethanol concentration of 25% (v/v).
  • LNP were prepared by standard NanoAssemblrTM BenchtopTM procedure.
  • the first stream included a therapeutic agent in acetate buffer.
  • the second stream included lipid particle-forming materials in a second solvent.
  • Suitable second solvents include solvents in which the cationic lipids are soluble and that are miscible with the first solvent.
  • Representative second solvents include aqueous ethanol 90%, or anhydrous ethanol.
  • Alternate second solvents include 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, and other alcohols.
  • the LNP mixture was diluted into RNAse free tubes containing three to forty volumes of stirred phosphate buffered saline (PBS) buffer, pH 7.4. Ethanol was removed through dialysis in PBS, pH 7, or using AmiconTM centrifugal filters (Millipore, USA) at 3000 RPM. Once the required concentration for testing was achieved, the particles were filter sterilized using 200 um filters in aseptic conditions. "Empty" LNPs were similarly produced, with the oligonucleotide absent from the first buffer solution.
  • PBS stirred phosphate buffered saline
  • Particle size is an important characteristic of LNP, because size dictates biodistribution in vivo. Particle size was determined by dynamic light scattering using a ZetaSizer Nano ZSTM (Malvern Instruments, UK).
  • RNA concentration and Encapsulation Efficiency were measured by a modified RiboGreen assay (Quant-IT- Ribogreen ® Assay kit, Invitrogen).
  • the RiboGreen ® dye is a fluorescent nucleic acid stain for quantitating intact RNA. RiboGreen ® assay provides RNA quantitation with minimal consumption of sample. Encapsulation efficiency is defined as the percentage of RNA protected within the LNP. Briefly, standards and samples were prepared in a 96 well plate with and without 1% Triton- X100, and incubated at 37°C for 15 minutes to break open the RNA LNPs. On completion of incubation, Ribogreen ® reagent was added to samples and standards, and fluorescence intensities of standards and samples with and without Triton-X100 were measured (Exc.
  • Percent Encapsulation or Encapsulation Efficiency is defined by the following equation:
  • Encapsulation Efficiency [(Total RNA - Free RNA (un-encapsulated RNA) Total RNA]%
  • Cortex tissue from E18 Sprague Dawley rat was purchased from BrainBits, LLC, Springfield, IL. This tissue was processed and the neuronal cells plated using the neuronal plating protocol from StemCell Technologies, Vancouver, BC. In short, the E18 cortex tissues were removed from the shipping medium and digested for 20 minutes using 0.25% trypsin. Following this, the trypsin was inactivated using DMEM media containing 10% FBS. The tissue was then pelleted and the FBS containing media was replaced with fresh DMEM.
  • the tissue was again pelleted and the pellet was triturated in Neuronal Culture Media supplemented with SMI (StemCell Technologies).
  • the cell suspension was then passed through a 40 ⁇ cell strainer to form a single cell suspension.
  • the concentration of the cell suspension was assessed using trypan blue and a hemocytometer.
  • the cell suspension was seeded at a density of 4.8 x 10 4 cells/cm 2 on PDL coated plates, or at a density of 3.2 x 10 4 cells/cm 2 on PDL coated coverslips.
  • the cells were incubated in a 37°C incubator with 5% C0 2 and half of the media was changed with fresh media every 3-4 days. Confirmation of neuronal cell type was done using visual inspection of the culture by microscope, as well as positive MAP2 staining. (MAP2 is a marker of neuronal cells).
  • neurons were treated with 2 ⁇ g/mL of mRNA LNP and supplemented with 5 ⁇ g/mL of ApoE (Peprotech Inc., USA). Best results were obtained when both mRNA and plasmid were added to human NPCs when they were seeded. Thus when the cells were passaged, they were seeded in individual wells and then the APoE and LNP added immediately. Forty-eight hours after being seeded, the neurons were treated with 2 ⁇ g/mL of mRNA LNP, and 24H after treatment, harvested for downstream assessment. Immunocytochemistry
  • Cells were seeded at the appropriate density on PDL coated coverslips. After treatment and incubation, the cells were fixed by 4% paraformaldehyde, permeabilized using 0.1% Triton X-100 in PBS, and then blocked using 10% normal donkey serum. Following blocking, primary antibody was added- MAP2 (Invitrogen) - and incubated overnight at 4°C. The following day, primary antibody was removed and secondary antibody was added as well as DAPI to stain the nucleus. The coverslips were then mounted on glass slides using ProLong DiamondTM fixative, (ThermoScientific, Waltham, MA). Images were acquired using a confocal microscope.
  • PrestoBlue ® cell viability reagent (ThermoScientific) was added, and the plate was incubated for 30 minutes at 37°C before being read using a plate reader (Synergy HI plate reader (BioTek, Winooski, VT)) using the reader specifications in the PrestoBlue ® cell viability reagent.
  • the cells were harvested following the protocol provided with SimpleStepTM GFP ELISA, AbCam. In short, the cells were washed with PBS and then lysed using the provided lysis buffer. Following lysis and collection the lysate was incubated on ice and then centrifuged to remove any remaining undigested cell debris. Total protein concentration was assessed using the BCA Protein Quantification Kit (AbCam, Cambridge, UK). The total protein concentration for each sample was then normalized to lng/ ⁇ for use in the SimpleStepTM GFP ELISA. EXAMPLE 1
  • Particle size (hydrodynamic diameter of the particles) was determined by Dynamic Light Scattering (DLS) using a ZetaSizer Nano ZSTM, Malvern Instruments, UK). He/Ne laser of 633nm wavelength was used as the light source. Data were measured from the scattered intensity data conducted in backscattering detection mode (measurement angle 173 °). Measurements were an average of 10 runs of two cycles each per sample. Z -Average size was reported as the LNP size, and is defined as the harmonic intensity averaged particle diameter. Nucleic acid encapsulation was measured using the Ribogreen ® dye method. A standard curve was established using known concentrations of control RNA sample, then test solutions were run and their measurements translated using the standard curve. Results are shown in Table 1.
  • Formulations contained various ratios of the cationic lipid l,17-bis(2- octylcyclopropyl)heptadecan-9-yl 4-(dimethylamino)butanoate hydrochloride, structural lipids selected from DSPC and DOPE, Myrj52 as a stabilizing agent, and 0.5MOL% of DiD.
  • Lipid Mix A Lipid Mix G and Lipid Mix H, cholesterol was present.
  • Lipid Mix J it was not.
  • Lipid Mix G Lipid Mix H, and Lipid Mix J, triglyceride was present.
  • Lipid Mix A and D triolein was not present. All of the formulations shown in Table 2 formed LNP with good size and PDI characteristics.
  • Lipid Mix G and Lipid mix D mRNA LNP with GFP were compared to industry standard Lipofectamine ® MessengerMaxTM transfection reagent under seven conditions at 48h:
  • Results are shown in Figure 1 as a bar graph of in vitro neuron viability demonstrated by PrestoBlue assay. Viability of neurons was well maintained by the Lipid Mix G and H LNP.
  • LNP Lipid Nanoparticles
  • NanoAssemblrTM Benchtop micromixer depending on the volume.
  • Uptake of HPRT siRNA was measured of Day 0, Day 2, Day 14 and Day 28. Knockdown of HPRT by said siRNA was measured at the same timepoints.
  • SiRNA LNP (1000, 100 and 10 ng/ml) along with a non-coding control formulation was used as a control when collecting the gene knockdown data.
  • LNP Formulations Lipid Mix A, Lipid Mix G, and Lipid Mix H containing EGFP mRNA (GFP) were tested on enriched neurons, and GFP expression was assessed by confocal microscopy, flow cytometry, and ELISA. Encapsulation efficiency was established for LNP as set out in Table 1. Fluorescence microscopy or confocal microscopy was used to identify GFP protein levels.
  • Figure 3 is a black and white, image reversed photograph of confocal microscopy of live neurons that have been treated with labeled Lipid Mix G LNP containing GFP.
  • the reverse image showed the results better than the original colour photograph for black and white image.
  • Both images include DAPI, a nuclear stain.
  • the rightmost image shows MAP2 (a neuronal marker) antibody staining, and the leftmost image is a merge showing
  • MAP2 antibody staining (a neuronal marker) shows the outline of the neurons.
  • DAPI staining of the successfully delivered mRNA to the nuclei is dark in both the left and the right panel.
  • GFP expression in the neurons is shown as even darker staining predominantly in the right panel. Not shown, no GFP expression was seen in neurons treated with Lipid Mix A mRNA LNP by the same confocal imaging process.
  • Lipid Mix G LNP was an effective mRNA delivery vehicle in live neurons.
  • the upper bar graph shows the percentage of neurons positive for GFP expression obtained by flow cytometry analysis after treatment with Lipid Mix A and G
  • the first bar graph in Figure 4 corresponds to the percentage of neurons positive for GFP, while the lower bar graph shows the mean fluorescent intensity of GFP expression in neurons treated with Lipid Mix A and Lipid Mix G nanoparticles (2.5 ug/mL of LNP + 5 ug/mL ApoE). Note how the expression of GFP is much greater for Lipid Mix G GFP LNP than Lipid Mix A GFP LNP.
  • the MFI result is a better measure of degree of expression of the delivered mRNA, suggesting that Lipid Mix A is an inferior carrier when it comes to mRNA expression, despite working well for siRNA delivery.
  • Lipid Mix A GFP LNP and Lipid Mix H GFP LNP are shown in Figure 5.
  • the upper bar graph in Figure 5 is the percentage of neurons with GFP present. Lipid Mix H gave better mRNA expression results, as shown in the lower MFI bar graph.
  • the results of ELISA analysis of the levels of GFP expression levels (pg/mL) in DIV7 neurons transfected with 2.5 ug/mL of LNP + 5 ug/mL ApoE are shown in Figure 6.
  • the four bars represent, respectively, the amount of GFP detected in a) untreated neurons, b) neurons treated with Lipid Mix A GFP LNP, c) neurons treated with Lipid Mix G GFP LNP, and d) neurons treated with Lipid Mix H GFP LNP.
  • Lipid Mix A performs little better than no treatment, while Lipid Mix G and H produced excellent GFP expression results.
  • Plasmid preparation pCMV-GFP 3487 bp, circular, double stranded DNA, SEQ ID. No. 6
  • Kanamycin resistant was ordered from PlasmidFactory, Germany, and stored in water for injection.
  • the plasmid included a GFP reporter gene which produces target protein only when the plasmid is successfully expressed within a cell.
  • the physical characteristics of plasmid LNP using Lipid Mix A, G, H and J are shown in Table 4. The size and PDI of the manufactured LNPs with Lipid Mix A, G, H and J were very similar.
  • EXAMPLE 8 Plasmid expression in vitro Lipid Mix A, Lipid Mix G, Lipid Mix H and Lipid Mix J LNP with pCMV-GFP plasmid encapsulated were added to cell cultures of neurons (DIV7) as described in Methods above, for 48 hours. Excellent expression was noted in the cell cultures treated with Lipid Mix G, Lipid Mix H and Lipid Mix J, as measured by mean fluorescence intensity (MFI) by flow cytometry. Results are shown in Figure 7.
  • MFI mean fluorescence intensity
  • Viability of the cells was not reduced by any of the formulations (as compared to untreated) for plasmid containing lipid nanoparticles comprised of Lipid Mix G, and Lipid Mix H at 1 ug/mL LNP and 5 ug/mL ApoE, as shown in Figure 8.
  • LNP comprised of Lipid Mix J is a cholesterol-free triolein formulation capable of transfecting neurons with plasmid DNA as shown in Figure 7.
  • GFP LNP GFP expression was quantified using the GFP ELISA protocol in Methods above.
  • mice were sacrificed and their spleens harvested. Spleen tissue tested for GFP levels. Results shown in pg/mL in Figure 9. Results are shown for Lipid Mix D LNP and Lipid Mix H LNP. The triolein-containing LNP gives substantially better expression of the mRNA than Lipid Mix D LNP. While specific embodiments of the invention have been described and illustrated, such embodiments should be considered illustrative of the invention only and not as limiting the invention as construed in accordance with the accompanying claims.

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Abstract

L'invention concerne une composition de réactif de transfection comprenant : 30 à 60 % en moles d'un lipide cationique, ou d'un sel pharmaceutiquement acceptable de celui-ci ; 10 à 60 % en moles d'un lipide structurel ; 10 à 20 % en moles d'un triglycéride ; et 0,1 à environ 10 % en moles d'un agent stabilisant. L'agent de transfection est efficace pour transfecter des cellules, en particulier des neurones, avec un ARNsi, un ARNm et un acide nucléique plasmidique, et maintenir la viabilité des cellules ainsi que l'activité de l'acide nucléique délivré.
PCT/CA2017/051543 2016-10-03 2017-12-19 Compositions pour la transfection de types de cellules résistantes WO2018119514A1 (fr)

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Cited By (6)

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WO2020210901A1 (fr) * 2019-04-15 2020-10-22 Precision Nanosystems Inc. Modification non virale de l'expression d'un gène de lymphocyte t
US11591544B2 (en) 2020-11-25 2023-02-28 Akagera Medicines, Inc. Ionizable cationic lipids
US12024484B2 (en) 2020-09-15 2024-07-02 Verve Therapeutics, Inc. Lipid formulations for gene editing
US12029795B2 (en) 2020-04-09 2024-07-09 Verve Therapeutics, Inc. Base editing of PCSK9 and methods of using same for treatment of disease
US12064479B2 (en) 2022-05-25 2024-08-20 Akagera Medicines, Inc. Lipid nanoparticles for delivery of nucleic acids and methods of use thereof
WO2025003754A1 (fr) * 2023-06-28 2025-01-02 Sanofi Utilisation de glycérides pour formulations de nanoparticules lipidiques

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WO2009127060A1 (fr) * 2008-04-15 2009-10-22 Protiva Biotherapeutics, Inc. Nouvelles formulations lipidiques pour l'administration d'acides nucléiques
WO2011000106A1 (fr) * 2009-07-01 2011-01-06 Protiva Biotherapeutics, Inc. Lipides cationiques et procédés améliorés pour l'administration d'agents thérapeutiques

Patent Citations (2)

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WO2009127060A1 (fr) * 2008-04-15 2009-10-22 Protiva Biotherapeutics, Inc. Nouvelles formulations lipidiques pour l'administration d'acides nucléiques
WO2011000106A1 (fr) * 2009-07-01 2011-01-06 Protiva Biotherapeutics, Inc. Lipides cationiques et procédés améliorés pour l'administration d'agents thérapeutiques

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020210901A1 (fr) * 2019-04-15 2020-10-22 Precision Nanosystems Inc. Modification non virale de l'expression d'un gène de lymphocyte t
CN113710811A (zh) * 2019-04-15 2021-11-26 精密纳米系统股份有限公司 T细胞基因表达的非病毒修饰
JP2022528996A (ja) * 2019-04-15 2022-06-16 プレシジョン ナノシステムズ インコーポレーテッド Ｔ細胞遺伝子発現の非ウイルス性改変
JP7447389B2 (ja) 2019-04-15 2024-03-12 プレシジョン ナノシステムズ ユーエルシー Ｔ細胞遺伝子発現の非ウイルス性改変
CN113710811B (zh) * 2019-04-15 2024-05-14 精密纳米系统无限责任公司 T细胞基因表达的非病毒修饰
US12029795B2 (en) 2020-04-09 2024-07-09 Verve Therapeutics, Inc. Base editing of PCSK9 and methods of using same for treatment of disease
US12115230B2 (en) 2020-04-09 2024-10-15 Verve Therapeutics, Inc. Base editing of ANGPTL3 and methods of using same for treatment of disease
US12024484B2 (en) 2020-09-15 2024-07-02 Verve Therapeutics, Inc. Lipid formulations for gene editing
US11591544B2 (en) 2020-11-25 2023-02-28 Akagera Medicines, Inc. Ionizable cationic lipids
US12077725B2 (en) 2020-11-25 2024-09-03 Akagera Medicines, Inc. Ionizable cationic lipids
US12064479B2 (en) 2022-05-25 2024-08-20 Akagera Medicines, Inc. Lipid nanoparticles for delivery of nucleic acids and methods of use thereof
WO2025003754A1 (fr) * 2023-06-28 2025-01-02 Sanofi Utilisation de glycérides pour formulations de nanoparticules lipidiques

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