Lipid nanoparticle formulation for central nervous system delivery

Abstract

For example, provided herein are compositions and methods for delivering nucleic acids such as therapeutic mRNA and DNA to the central nervous system by using sphingolipids as helper lipids to deliver lipid nanoparticles containing the nucleic acids into the subarachnoid space or the brain.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 338,216, filed on May 4, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Lipid nanoparticles are efficient carriers for cargos such as nucleic acid cargos for delivery into cells, particularly in applications such as gene delivery, mRNA delivery, antisense, and RNA interference. Lipid nanoparticles typically comprise helper lipids, cholesterol, ionizable lipids (e.g., lipidoids), lipid-polymer conjugates, and nucleic acid cargos. Lipid nanoparticles are typically administered by intravenous, intramuscular, or subcutaneous injection. Exemplary LNP compositions and / or compositions useful in the manufacture of LNPs, such as lipidoids, are described in U.S. Patent Nos. 10,844,028; 10,189,802; 9,872,911; 9,556,110; 9,439,968; 9,227,917; 8,969,353; and 8,450,298, and U.S. Patent Application Publication Nos. 2017 / 0204075; 2019 / 0177289; 2017 / 0152213; 2016 / 0114042; 2015 / 0203439; 2014 / 0322309; 2014 / 0161830; 2011 / 0293703; and 2010 / 0331234, each of which is incorporated herein by reference to the extent that the technical disclosures related to compounds and compositions useful for the delivery of nucleic acid cargos are consistent with the present disclosure.Further examples of lipid nanoparticles are described in U.S. Patent Nos. 9,404,127, 9,364,435, and 8,058,069, each of which is incorporated herein by reference in the foregoing scope consistent with the present disclosure for the technical disclosures related to compounds and compositions useful for the delivery of nucleic acid cargo (see, e.g., Sabnis S, et al., A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates. Mol Ther. 2018;26(6):1509-1519 and Yonezawa S, et al., Recent advances in siRNA delivery mediated by lipid-based nanoparticles. Adv Drug Deliv Rev. 2020;154-155:64-78). Examples of the lipid nanoparticles, lipidoids, and methods of making the lipid nanoparticles and lipidoids described herein are described in Whitehead KA, et al., Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity. Nat Commun. 2014 Jun 27;5:4277. doi:10.1038 / ncomms5277. PMID:24969323; PMCID:PMC4111939.

Prior Art Documents

Patent Documents

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Non-Patent Document

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Non-Patent Document 1

[0005] Although successful in the parenteral delivery of nucleic acids via LNPs, there are significant obstacles to the delivery of nucleic acids to cells of the central nervous system (CNS). A vehicle for effectively delivering nucleic acids to the cells of the central nervous system is needed. [Means for Solving the Problems]

[0006] [Summary of the Invention] According to a first embodiment or aspect of the invention, a method of delivering a therapeutic agent to tissue of the central nervous system (CNS) of a patient is provided. The method includes administering to the tissue of the CNS of the patient a composition comprising lipid-containing particles comprising a therapeutic agent, the lipid-containing particles further comprising a sphingolipid helper lipid; cholesterol or a derivative thereof; a PEG-based compound such as a PEG-containing polymer or a PEGylated fatty acid-containing compound; and an ionizable lipidoid.

[0007] According to a further embodiment or aspect of the invention, lipid-containing particles are provided. The lipid-containing particles comprise a therapeutic agent, and the lipid-containing particles further comprise a sphingolipid helper lipid; cholesterol or a derivative thereof; a PEG-based compound such as a PEG-containing polymer or a PEGylated fatty acid-containing compound; and an ionizable lipidoid.

[0008] The following numbered items outline various aspects or embodiments of the invention.

[0009] Item 1 A method of delivering a therapeutic agent to tissue of the central nervous system (CNS) of a patient, the method comprising administering to the tissue of the CNS of the patient a composition comprising lipid-containing particles comprising a therapeutic agent, the lipid-containing particles further comprising a sphingolipid helper lipid; cholesterol or a derivative thereof; a PEG-based compound such as a PEG-containing polymer or a PEGylated fatty acid-containing compound; and an ionizable lipidoid.

[0010] Item 2 The method according to item 1, wherein the lipid-containing particles are lipid nanoparticles.

[0011] Item 3 The method according to item 1 or 2, wherein the lipid-containing particles are administered into the subarachnoid space, into the brain, or to the brainstem of the patient.

[0012] Item 4 The method according to any one of items 1 to 3, wherein the sphingolipid is ceramide, sphingomyelin, ceramide phosphoethanolamine, sphingosylphosphorylcholine, sphingosine, sphingoglycolipid, or any combination of two or more thereof.

[0013] Item 5. The method according to item 4, wherein the sphingolipid is ceramide, glucosylceramide, galactosylceramide, or lactosylceramide.

[0014] Item 6. The method according to any one of items 1 to 5, wherein the sphingolipid is a CNS sphingolipid, a brain sphingolipid, ceramide, lactosylceramide, galactosylceramide, glucosylceramide, or a combination of any two or more thereof.

[0015] Item 7. The method according to any one of items 1 to 5, wherein the sphingolipid is ceramide, lactosylceramide, galactosylceramide, or a combination of any two or more thereof.

[0016] Item 8. The lipidoid is 306 Oi10 ; 306O 10 ; 503O i10 ; 402O 6,10 ; 500X 1 ; 500O i10 ; 306O 11 ; 306O i10 ; 306O 12 ; 200X 6 ; 516O i10 ; 500O 1,1,8 ; 514X 6 ; 306O 14 ; 501X 1 ; 205O 16 ; 500O 13 ; 113O i10 ; 306O 16 ; 306O 13 ; 205O 18 ; 509X 7 ; 501O i10 ; 503O i10 ; 500O 14 ; 113O i10 ; 509X 1 ; 509X 3 ; 501X 2 ; 402O 6,10 ; 516O 4,8 ; 402X8 ; 501O 1,1,8 ; or 509O 1,1,8 The method according to any one of items 1 to 7, which is one or more of the above.

[0017] Item 9 The lipidoid is 306 Oi10 , 306O 10 503O i10 and 402O 6,10 The method according to any one of items 1 to 7, which is one or more of the above.

[0018] Item 10 The ionizable lipidoid is 306 Oi10 The method according to item 1.

[0019] Item 11 The therapeutic agent is anionic or polyanionic. The method according to any one of items 1 to 10.

[0020] Item 12 The therapeutic agent is a nucleic acid. The method according to any one of items 1 to 10.

[0021] Item 13 The nucleic acid contains RNA. The method according to item 12.

[0022] Item 14 The RNA contains mRNA. The method according to item 13.

[0023] Item 15 The RNA contains an RNAi reagent, dsRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA (gRNA), long non-coding RNA (lncRNA), base editing gRNA (beRNA), prime editing gRNA (pegRNA), or transfer RNA (tRNA). The method according to item 13.

[0024] Item 16 The RNA contains gRNA, beRNA, or pegRNA and mRNA encoding Cas9 for base editing or prime editing, or a Cas9 fusion protein. The method according to item 13.

[0025] Item 17. The method according to item 12, wherein the nucleic acid encodes heme oxygenase 1 (HO1), brain-derived neurotrophic factor (BDNF), or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein.

[0026] Item 18. The method according to item 12, wherein the nucleic acid is DNA.

[0027] Item 19. The lipid-containing particles are lipid nanoparticles comprising, in mol%, 5 to 95 mol% of the sphingolipid helper lipid, 5 to 75 mol% of the cholesterol or its derivative, 0.1 to 50 mol% of the PEG-based compound, and 5 to 90 mol% of the ionizable lipidoid, according to any one of items 1 to 17.

[0028] Item 20. The method according to any one of items 1 to 19, wherein the cholesterol or its derivative is cholesterol.

[0029] Item 21. The method according to any one of items 1 to 20, wherein the PEG-based compound is one or more of PEG-ceramide, PEG-DMG, PEG-PE, poloxamer, or DSPE carboxy PEG. For example, in certain embodiments, the PEG-based substance is C14PEG2000DMG, C15PEG2000DMG, C16PEG2000DMG, C18PEG2000DMG, C14PEG2000 ceramide, C15PEG2000 ceramide, C16PEG2000 ceramide, C18PEG2000 ceramide, C14PEG2000PE, C15PEG2000PE, C16PEG2000PE, C18PEG2000PE, C14PEG350PE, C14PEG5000PE, poloxamer F-127, poloxamer F-68, poloxamer L-64, and DSPE carboxy PEG.

[0030] Item 22. The PEG-based compound is a PEGylated fatty acid, such as PEGylated C 10 -C 20A fatty acid-containing compound, such as C 14 -PEG 2000 -PE, the method according to item 21.

[0031] Item 23 The method according to any one of items 1 to 22, wherein the nucleic acid encodes heme oxygenase 1 (HO1) and / or brain-derived neurotrophic factor (BDNF) for the treatment of ischemia or ischemia / reperfusion injury, such as ischemic stroke, in the patient.

[0032] Item 24 The method according to any one of items 1 to 22, wherein the nucleic acid encodes a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein for the treatment of cancer, such as glioblastoma, in the patient.

[0033] Item 25 A lipid-containing particle comprising a therapeutic agent, further comprising a sphingolipid helper lipid; cholesterol or a derivative thereof; a PEG-based compound such as a PEG-containing polymer or a PEGylated fatty acid-containing compound; and an ionizable lipidoid, the lipid-containing particle.

[0034] Item 26 The lipid-containing particle according to item 25, wherein the lipid-containing particle is a lipid nanoparticle.

[0035] Item 27 The lipid-containing particle according to item 25 or 26, wherein the sphingolipid is ceramide, sphingomyelin, ceramide phosphoethanolamine, sphingosylphosphorylcholine, sphingosine, sphingoglycolipid, or any combination of two or more thereof.

[0036] Item 28 The lipid-containing particle according to item 27, wherein the sphingolipid is ceramide, glucosylceramide, galactosylceramide, or lactosylceramide.

[0037] Item 29. The lipid-containing particles according to any one of Items 25 to 28, wherein the sphingolipid is a CNS sphingolipid, a brain sphingolipid, a ceramide, a lactosylceramide, a galactosylceramide, a glucosylceramide, or a combination of any two or more thereof.

[0038] Item 30. The lipid-containing particles according to any one of Items 25 to 28, wherein the sphingolipid is a ceramide, a lactosylceramide, a galactosylceramide, or a combination of any two or more thereof.

[0039] Item 31. The lipidoid is 306 Oi10 ; 306O 10 ; 503O i10 ; 402O 6,10 ; 500X 1 ; 500O i10 ; 306O 11 ; 306Oi10; 306O 12 ; 200X 6 ; 516O i10 ; 500O 1,1,8 ; 514X 6 ; 306O 14 ; 501X 1 ; 205O 16 ; 500O 13 ; 113O i10 ; 306O 16 ; 306O 13 ; 205O 18 ; 509X 7 ; 501O i10 ; 503O i10 ; 500O 14 ; 113O i10 ; 509X 1 ; 509X 3 ; 501X 2 ; 402O 6,10 ; 516O 4,8 ; 402X 8 ; 501O 1,1,8 ; or 509O 1,1,8 The lipid-containing particles according to any one of Items 25 to 30, which are one or more of the above.

[0040] Item 32. The lipidoid is 306Oi10 、306O 10 、503O i10 、and 402O 6,10 The lipid-containing particle according to any one of items 25 to 30, which is one or more of the above.

[0041] Item 33 The ionizable lipidoid is 306 Oi10 The lipid-containing particle according to item 25, wherein the ionizable lipidoid is 306.

[0042] Item 34 The therapeutic agent is anionic or polyanionic. The lipid-containing particle according to any one of items 25 to 33.

[0043] Item 35 The therapeutic agent is a nucleic acid. The lipid-containing particle according to any one of items 25 to 33.

[0044] Item 36 The nucleic acid contains RNA. The lipid-containing particle according to item 35.

[0045] Item 37 The RNA contains mRNA. The lipid-containing particle according to item 36.

[0046] Item 38 The RNA contains an RNAi reagent, dsRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA (gRNA), long non-coding RNA (lncRNA), base editing gRNA (beRNA), prime editing gRNA (pegRNA), or transfer RNA (tRNA). The lipid-containing particle according to item 36.

[0047] Item 39 The RNA contains a gRNA, beRNA, or pegRNA, and an mRNA encoding Cas9 for base editing or prime editing, or a Cas9 fusion protein. The lipid-containing particle according to item 36.

[0048] Item 40 The nucleic acid encodes heme oxygenase 1 (HO1), brain-derived neurotrophic factor (BDNF), or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein. The lipid-containing particle according to item 35.

[0049] Item 41. The lipid-containing particle according to item 35, wherein the nucleic acid is DNA.

[0050] Item 42. The lipid-containing particle according to any one of items 25 to 41, wherein the lipid-containing particle is a lipid nanoparticle containing 5 to 95 mol% of the sphingolipid helper lipid, 5 to 75 mol% of the cholesterol or its derivative, 0.1 to 50 mol% of the PEG-based compound, and 5 to 90 mol% of the ionizable lipidoid in terms of mol% of the sphingolipid helper lipid, the cholesterol or its derivative, the PEG-based compound, and the lipidoid.

[0051] Item 43. The lipid-containing particle according to any one of items 25 to 42, wherein the cholesterol or its derivative is cholesterol.

[0052] Item 44. The lipid-containing particle according to any one of items 25 to 43, wherein the PEG-based compound is one or more of PEG-ceramide, PEG-DMG, PEG-PE, poloxamer, or DSPE carboxy PEG. For example, in certain embodiments, the PEG-based substance is C14PEG2000DMG, C15PEG2000DMG, C16PEG2000DMG, C18PEG2000DMG, C14PEG2000 ceramide, C15PEG2000 ceramide, C16PEG2000 ceramide, C18PEG2000 ceramide, C14PEG2000PE, C15PEG2000PE, C16PEG2000PE, C18PEG2000PE, C14PEG350PE, C14PEG5000PE, poloxamer F-127, poloxamer F-68, poloxamer L-64, and DSPE carboxy PEG.

[0053] Item 45. The lipid-containing particle according to item 44, wherein the PEG-based compound contains a PEGylated fatty acid, for example, PEGylated C 10 -C 20 a fatty acid-containing compound, for example, C 14 -PEG 2000 -PE.

[0054] Item 46. The lipid-containing particle according to any one of Items 25 to 45, wherein the nucleic acid encodes heme oxygenase 1 (HO1) and / or brain-derived neurotrophic factor (BDNF) for the treatment of ischemia or ischemia / reperfusion injury in the patient, such as ischemic stroke.

[0055] Item 47. The lipid-containing particle according to any one of Items 25 to 45, wherein the nucleic acid encodes a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein for the treatment of cancer in the patient, such as glioblastoma.

[0056] Item 48. The lipid-containing particle according to any one of Items 25 to 45 for the treatment of neuroinflammation, neurodegenerative disease, or monogenic neuropathy.

[0057] Item 49. A method of treating a patient suffering from ischemia, stroke, ischemia / reperfusion injury, neuroinflammation, neurodegenerative disease, monogenic neuropathy, or cancer, the method comprising administering to the patient an effective amount of the lipid nanoparticles according to any one of Items 25 to 45, thereby treating the ischemia, stroke, ischemia / reperfusion injury, neuroinflammation, neurodegenerative disease, monogenic neuropathy, or cancer in the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0058]

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Mode for Carrying Out the Invention

[0059] Unless there are operating examples or separate instructions, the use of numerical values within the various ranges specified in this application is described as approximate values as if the word "about" preceded both the minimum and maximum values within the stated range. Thus, values that vary slightly above and below the stated range can be used to achieve substantially the same results as the values within the range. Also, unless otherwise indicated, the disclosure of a range is intended as a continuous range that includes all values between the minimum and maximum values. A range between two numerical values, including these two numerical values, can alternatively be described as a range from the first numerical value to the second numerical value. For example, "n ranges from a to b, including both ends" can be rephrased as "n is from a to b". The terms "more than" or "less than" can be used to exclude the stated value. For example, "n ranges from more than a to b" excludes a but includes b (i.e., a < n ≤ b).

[0060] As used herein, "a" and "an" refer to one or more.

[0061] The term "comprising" is open-ended and can be synonymous with "including", "containing", or "characterized by". The term "consisting essentially of" limits the claim to those that do not substantially affect the basic and novel features of the claimed invention by the specified substances or steps. The term "consisting of" excludes any element, step, or component not specified in the claim. As used herein, embodiments "comprising" one or more of the recited elements or steps include, but are not limited to, embodiments "consisting essentially of" and embodiments "consisting of" the recited elements or steps. With respect to the definitions presented herein, these definitions refer to the word forms, analogs, and grammatical variations of these words or phrases.

[0062] As used herein, the terms "patient" or "subject" refer to members of the animal kingdom, including but not limited to humans, and "mammal" refers to all mammals, including but not limited to humans.

[0063] "Treatment" in the context of a disease or disorder, a marker of a disease or disorder, or a symptom of a disease or disorder may refer to a clinically relevant and / or statistically significant decrease or increase in a confirmed value of a clinically relevant marker that moves from outside the normal range towards or reaches the normal range. The decrease or increase may be at least 10%, at least 20%, at least 30%, at least 40%, or more, for example, to a level that is acceptable as a treatment goal or within the normal range for an individual without such a disease or disorder, or, when decreasing the value, to a level below the detection level of the assay. The decrease or increase may be to a level that is acceptable within the normal range for an individual without such a disease or disorder, and may also be referred to as normalization of the level. The decrease or increase may be normalization of the level of a sign or symptom of a disease or disorder (i.e., a decrease in the difference between the subject level of the sign of the disease or disorder and the normal level of the sign of the disease or disorder), for example, up to the normal upper limit level if the value of the subject must be decreased to reach the normal level, and up to the normal lower limit level if the value of the subject must be increased to reach the normal level.

[0064] The compositions described herein may contain, as active agents, nucleic acid reagents such as, but not limited to, DNA, RNA (e.g., mRNA), antisense reagents, or RNAi (RNA interference) reagents.

[0065] As used herein, the term "cell" refers to any type of cell from any animal, such as, but not limited to, rats, mice, monkeys, and humans. For example, but not limited to, cells can be progenitor cells, such as stem cells, induced pluripotent stem cells, pluripotent cells including multipotent cells, or differentiated cells such as endothelial cells and smooth muscle cells. "Cells" can be, for example, in vivo as part of a tissue or organ, or can be in vitro, such as, but not limited to, a population of cells rich in a particular cell type, such as progenitor cells or stem cells.

[0066] A composition is "biocompatible" in that the composition and, if applicable, its components, or its degradation products, are substantially non-toxic to cells or organisms within acceptable limits, such as being substantially non-carcinogenic and substantially non-immunogenic, and are removed from a biological system, such as a living organism (patient), without substantial toxic effects or, alternatively, degrade. Non-limiting examples of degradation mechanisms within a biological system include chemical reactions, hydrolysis reactions, and enzymatic cleavage.

[0067] A "therapeutically effective amount", as used herein, can include an amount of a lipid-containing particle, such as an LNP described herein, that can be sufficient to effect treatment of a disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of a disease) when administered to a subject afflicted with the disease. A "therapeutically effective amount" can vary depending on the lipid-containing particle, such as an LNP, how the composition is administered, the disease and its severity, and the medical history, age, weight, family history, genetic makeup of the subject being treated, the type of prior or concurrent treatment, if any, and other individual characteristics.

[0068] "Therapeutically effective amount" can also include an amount of an agent that produces a local or systemic effect at a reasonable risk-to-benefit ratio applicable to any treatment. Lipid-containing particles such as LNPs used in the methods described herein can be administered in an amount sufficient to produce a reasonable risk-to-benefit ratio applicable to such treatment.

[0069] As used herein, the phrase "pharmaceutically acceptable carrier" may refer to a liquid or solid filler, diluent, excipient, manufacturing aid (such as a lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or a solvent encapsulating material, etc., which is a pharmaceutically acceptable material, composition or vehicle involved in the transport or conveyance of the subject compound from one organ or part of the body to another organ or another part of the body. Each carrier can be "acceptable" in the sense that it is compatible with the other components of the formulation and not harmful to the subject being treated. Some non-limiting examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; and (24) other non-toxic compatible substances used in pharmaceutical formulations.

[0070] "Group" or "functional group" refers to a part of a larger molecule that contains or consists of a collection of atoms and / or bonds that impart chemical or physical properties to the molecule. "Residue" refers to the portion of a compound or monomer that remains in a larger molecule, such as a polymer chain, after the compound or monomer has been incorporated into the larger molecule. "Moiety" refers to a part of a molecule that may contain one or more functional groups and, in the case of an "active moiety", may be the characteristic part of a molecule or compound that imparts activity, such as pharmacological or physiological activity, as contrasted with an inactive part of the molecule, such as an ester of the active moiety or a salt of the active agent.

[0071] As used herein, the term "polymer composition" refers to a composition that contains one or more polymers. As a class, "polymers" include, but are not limited to, homopolymers, heteropolymers, copolymers, block polymers, block copolymers, and may be both natural and synthetic. A homopolymer contains one type of component, or monomer, while a copolymer contains more than one type of monomer.

[0072] A polymer "contains" or is "derived from" a monomer if the stated monomer is incorporated into the polymer. Thus, the incorporated monomer contained in the polymer is not the same as the monomer prior to incorporation into the polymer in that at least certain groups, such as end groups that are modified during polymerization, may be changed, removed, and / or rearranged, and certain bonds may be added, removed, and / or modified when the monomer is incorporated. A polymer is said to contain a particular bond if that bond is present in the polymer. Unless otherwise specified, the molecular weight of a polymer composition refers to the weight average molecular weight (M W ).

[0073] As used herein, "alkyl" refers to, for example, a straight-chain, branched-chain, or cyclic hydrocarbon group containing from 1 to about 20 carbon atoms, such as, but not limited to, C 1-3 , C 1-6 , C 1-10The radical, for example but not limited to, refers to straight-chain or branched-chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc. The alkyl group can be, for example, substituted or unsubstituted, C 1 -C 2 -C 3 -C 4 -C 5 -C 6 -C 7 -C 8 -C 9 -C 10 -C 11 -C 12 -C 13 -C 14 -C 15 -C 16 -C 17 -C 18 -C 19 -C 20 -C 21 -C 22 -C 23 -C 24 -C 25 -C 26 -C 27 -C 28 -C 29 -C 30 -C 31 -C 32 -C 33 -C 34 -C 35 -C 36 -C 37 -C 38 -C 39 -C 40 -C 41 -C 42 -C 43 -C 44 -C 45 -C 46 -C 47 -C 48 -C 49 - or C 50 -radical. "Lower alkyl" refers to C 1 -C 6Refers to alkyl. Non-limiting examples of straight-chain alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Branched alkyl groups include any straight-chain alkyl group substituted with any number of alkyl groups. Non-limiting examples of branched alkyl groups include isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. Non-limiting examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. Cyclic alkyl groups also include fused, bridged, and spiro bicyclic, as well as higher-order fused, bridged, and spiro systems. Cyclic alkyl groups may be substituted with any number of straight-chain, branched, or cyclic alkyl groups. "Unsaturated alkyl" may contain one or more, for example, 1, 2, 3, 4, or 5 carbon-carbon double bonds, or may be referred to as alkene or alkenyl as described below. "Substituted alkyl" may include alkyl substituted at one or more (e.g., 1, 2, 3, 4, 5, 6, or more) positions, and this substituent is bonded to any available atom with the substitutions described herein to form a stable compound. "Optionally substituted alkyl" refers to alkyl or substituted alkyl. "Halogen", "halide", and "halo" refer to -F, -Cl, -Br, and / or -I. "Alkylene" and "substituted alkylene" include divalent alkyl and divalent substituted alkyl, respectively, and include, but are not limited to, methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nonamethylene, or decamethylene. "Optionally substituted alkylene" may include alkylene or substituted alkylene.

[0074] "Alkene or alkenyl" may include, for example, a straight-chain, branched-chain, or cyclic hydrocarbyl group containing from 2 to about 20 carbon atoms, and, for example, in the case of fatty acids, C 6-24may contain a base and have one or more, for example, 1, 2, 3, 4, or 5 carbon-carbon double bonds, and may be referred to as "unsaturated alkyl" in the context of fatty acids and lipids. The olefins of the alkenyl group can be, for example, E, Z, cis, trans, terminal, or exo-methylene. The alkenyl or alkenylene group is, for example, substituted or unsubstituted, C 2 、C 3 、C 4 、C 5 、C 6 、C 7 、C 8 、C 9 、C 10 、C 11 、C 12 、C 13 、C 14 、C 15 、C 16 、C 17 、C 18 、C 19 、C 20 、C 21 、C 22 、C 23 、C 24 、C 25 、C 26 、C 27 、C 28 、C 29 、C 30 、C 31 、C 32 、C 33 、C 34 、C 35 、C 36 、C 37 、C 38 、C 39 、C 40 、C 41 、C 42 、C 43 、C 44 、C 45 、C 46 、C 47 、C 48 、C 49 、or C 50It can be a radical. A haloalkenyl group can be any alkenyl group substituted with any number of halogen atoms. A "substituted alkene" can include an alkene substituted at one or more positions, for example, 1, 2, 3, 4, or 5 positions, and this substituent is bonded to any available atom by substitution as described herein to form a stable compound. An "optionally substituted alkene" can include an alkene or a substituted alkene. Similarly, "alkenylene" can refer to a divalent alkene. Examples of alkenylene include, but are not limited to, ethenylene (-CH=CH-) and all of its stereoisomers and conformational isomers. A "substituted alkenylene" can refer to a divalent substituted alkene. An "optionally substituted alkenylene" can refer to an alkenylene or a substituted alkenylene.

[0075] "Ester" is represented by the formula -OC(O)R, where R can be alkyl, alkenyl, or the above groups.

[0076] Alkyne or "alkynyl" refers to a straight-chain, branched-chain, or cyclic unsaturated hydrocarbon having the indicated number of carbon atoms and at least one triple bond. The triple bond of an alkyne or alkynyl group can be internal or terminal. (C 2 -C 8 ) Examples of alkynyl groups include, but are not limited to, acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne, 2-heptyne, 3-heptyne, 1-octyne, 2-octyne, 3-octyne, and 4-octyne. An alkynyl group can be unsubstituted or optionally substituted with one or more substituents as described herein below. An alkyne or alkynyl group is, for example, substituted or unsubstituted, C 2 、C 3 、C 4 、C 5 、C 6 、C 7 、C 8 、C 9 、C 10 、C 11, C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , C 25 , C 26 , C 27 , C 28 , C 29 , C 30 , C 31 , C 32 , C 33 , C 34 , C 35 , C 36 , C 37 , C 38 , C 39 , C 40 , C 41 , C 42 , C 43 , C 44 , C 45 , C 46 , C 47 , C 48 , C 49 , or C 50 may be a group. A haloalkynyl group can be any alkynyl group substituted with any number of halogen atoms. The term "alkynylene" refers to a divalent alkyne. Examples of alkynylene include, but are not limited to, ethynylene and propynylene. "Substituted alkynylene" refers to a divalent substituted alkyne.

[0077] "PEG" refers to polyethylene glycol. "PEGylation" refers to a compound containing a moiety that includes two or more contiguous ethylene glycol moieties. Non-limiting examples of PEG moieties for PEGylation of a compound include -(O-CH 2 -CH 2 ) n -, -(CH 2 -CH 2 -O) n , or -(O-CH 2 -CH 2 )n It contains one or more blocks of 1 to 200 ethylene glycol units such as -OH, and n is, for example but not limited to, in the range of 1 to 200 or 1 to 100, for example 1 to 5, or 1.

[0078] "Aryl" refers to an aromatic ring system such as phenyl or naphthyl, alone or in combination. "Aryl" may optionally include an aromatic ring system that may be fused with a cycloalkyl ring. "Substituted aryl" is an aryl independently substituted with one or more substituents bonded to any available atom to form a stable compound, and the substituents are as described herein. The substituents can be, for example, hydrocarbyl groups, alkyl groups, alkoxy groups, and halogen atoms. "Optionally substituted aryl" refers to aryl or substituted aryl. An aryloxy group can be, for example, an oxygen atom substituted with any aryl group such as phenoxy. An arylalkoxy group can be, for example, an oxygen atom substituted with any aralkyl group such as benzyloxy.

[0079] "Arylene" refers to divalent aryl, and "substituted arylene" refers to divalent substituted aryl.

[0080] "Optionally substituted arylene" refers to arylene or substituted arylene. Related terms such as "polycyclic aryl group" and "polycyclic aromatic group" refer to groups composed of at least two fused aromatic rings. "Heteroaryl" or "hetero-substituted aryl" refers to an aryl group substituted with one or more heteroatoms such as N, O, P, and / or S. Examples of heteroaryl groups include, but are not limited to, thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.

[0081] "Cycloalkyl" refers to a monocyclic, bicyclic, tricyclic, or polycyclic 3- to 14-membered ring system, which can be either saturated or partially unsaturated. The cycloalkyl group may be attached via any atom. Cycloalkyl also encompasses fused rings where the cycloalkyl is fused to an aryl or heteroaryl ring. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The cycloalkyl group may be unsubstituted or optionally substituted with one or more substituents described hereinafter in this specification. "Cycloalkylene" refers to a divalent cycloalkyl. The term "optionally substituted cycloalkylene" refers to a cycloalkylene substituted with at least 1, 2, or 3 substituents and attached to any available atom to form a stable compound, and the substituents are as described in this specification.

[0082] "Carboxyl" or "carboxylic", when so designated, refers to a group having the specified number of carbon atoms and terminated with a -C(O)OH group, and thus having the structure -R-C(O)OH, where R is an unsubstituted or substituted divalent organic group that may include linear, branched, or cyclic hydrocarbons. Non-limiting examples of these include carboxylic acid groups such as ethanoic acid, propanoic acid, 2-methylpropanoic acid, butanoic acid, 2,2-dimethylpropanoic acid, pentanoic acid, etc. 1-8 "Amine" or "amino", when so designated, refers to a group having the specified number of carbon atoms and terminated with a -NH 2 group, and thus having the structure -R-NH 2 . Here, R is an unsubstituted or substituted divalent organic group that may include, for example, linear, branched, or cyclic hydrocarbons and may optionally include one or more heteroatoms. The term "alkylamino" refers to a radical of the formula -NHR x or -NR x R x , where each R x is independently a defined alkyl radical.

[0083] Terms that combine the above refer to any suitable combination of the above, such as arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, and alkynylheteroaryl. As an example, "arylalkylene" refers to a divalent alkylene in which one or more hydrogen atoms in the alkylene group are substituted with an aryl group such as a (C 3 -C 8 )aryl group. Examples of the (C 3 -C 8 )aryl-(C 1 -C 6 )alkylene group include, but are not limited to, 1-phenylbutylene, phenyl-2-butylene, 1-phenyl-2-methylpropylene, phenylmethylene, phenylpropylene, and naphthylethylene. "(C 3 -C 8 )cycloalkyl-(C 1-C 6 ) The term "alkylene" refers to C 1 -C 6 One or more hydrogen atoms in the alkylene group are (C 3 -C 8 ) It refers to a divalent alkylene substituted with a cycloalkyl group. (C 3 -C 8 ) Examples of cycloalkyl-(C 1 -C 6 ) alkylene groups include, but are not limited to, 1-cyclopropylbutylene, cyclopropyl-2-butylene, cyclopentyl-1-phenyl-2-methylpropylene, cyclobutylmethylene, and cyclohexylpropylene.

[0084] Fatty acids are aliphatic monocarboxylic acids containing a carboxyl group bonded to an aliphatic hydrocarbyl group, which may be saturated or unsaturated. A hydrocarbyl or hydrocarbon group refers to a group of carbon and hydrogen atoms such as an alkyl, alkenyl (or unsaturated alkyl), or aryl group. "Aliphatic" means an acyclic or cyclic, saturated or unsaturated hydrocarbon compound excluding aromatic compounds. The aliphatic group of a fatty acid is typically a straight chain of carbon, but fatty acids and substituted fatty acids as a class include straight, branched, and / or cyclic carbon chains. As used herein, fatty acids include both natural aliphatic carboxylic acids and synthetic aliphatic carboxylic acids. Fatty acids can have an aliphatic chain of 3 to 40 carbon atoms (e.g., as used herein, "(C 3 -C 40 ) fatty acid"). Substituting a hydrogen atom of a compound such as a fatty acid with a group or moiety (hereinafter referred to as a "substituent") can yield a substituted fatty acid. Fatty acids and substituted fatty acids are sometimes referred to as "optionally substituted fatty acids". Fatty acids, and fatty acid groups, can be denoted by the number of carbon atoms and the number of double bonds. For example, C10:0 refers to a fatty acid or fatty acid group having 10 carbon atoms and 0 double bonds. Similarly, C18:1 refers to a fatty acid having an 18-carbon chain with one double bond, such as oleic acid.

[0085] Unsaturated fatty acids and substituted unsaturated fatty acids (collectively referred to herein as "optionally substituted unsaturated fatty acids") contain one or more carbon-carbon double bonds or alkenyl groups (e.g., vinyl groups) in their aliphatic chains. Each carbon atom of the alkenyl group is referred to herein as an alkenyl carbon. Unless otherwise specified, any carbon-carbon double bond in the alkyl chain of an optionally substituted unsaturated fatty acid can independently be an E (trans) or Z (cis) geometric isomer, or a mixture thereof.

[0086] Fatty acids can include, but are not limited to, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, and C40 fatty acids. The fatty acids can be saturated (without double bonds) or unsaturated, e.g., without double bonds or having 1, 2, 3, 4, 5, 6, or more double bonds. Non-limiting examples of saturated fatty acids include propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, carnoceric acid, montanic acid, nonacosylic acid, melissic acid, hentriacontylic acid, lacceroic acid, psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid, heptatriacontylic acid, octatriacontylic acid, nonatriacontylic acid, and tetracontylic acid.Non-limiting examples of unsaturated fatty acids include crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, nervonic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, eleostearic acid, mead acid, dihomo-γ-linolenic acid, eicosatrienoic acid, stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, osbond acid, sardine acid, tetracosanolpentaenoic acid, cervonic acid, and nisinic acid.

[0087] The compounds described herein, including fatty acids and substituted fatty acids, may exist in various isomeric forms, including configurational isomers, geometric isomers, and conformational isomers, and may also exist in various tautomeric forms, such as those with different attachment points of hydrogen atoms. The term "isomers" is intended to include all isomeric forms of the compounds of the present invention, including the tautomeric forms of said compounds.

[0088] Certain compounds described herein may have chiral centers and thus exist in different enantiomeric and diastereomeric forms. The compounds can be in the form of optical isomers or diastereomers. Accordingly, the compounds described herein include their optical isomers, diastereoisomers, and mixtures thereof, including racemic mixtures, unless otherwise specified. The optical isomers of the compounds of the present invention can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, simulated moving bed technology, or by chemical separation of stereoisomers using an optically active resolving agent.

[0089] Unless otherwise indicated, the term "stereoisomer" means one stereoisomer of a compound that substantially excludes other stereoisomers of the compound. Thus, a stereochemically pure compound having one chiral center substantially excludes the opposite enantiomer of the compound. A stereochemically pure compound having two chiral centers substantially excludes other diastereomers of the compound. A typical stereochemically pure compound contains more than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example, more than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, or more than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, or more than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound.

[0090] Lipids as a group include glycerides and phospholipids. "Glyceride" is an ester of glycerol (propane 1,2,3-triol) with a fatty acid or a substituted fatty acid. A phospholipid is a lipid containing phosphoric acid as a monoester or diester, such as phosphatidic acid and phosphoglyceride. A phosphoglyceride is a diester of glycerol, in which one hydroxyl group of the glycerol is phosphodiester-bonded to a group such as a functional group such as, for example, but not limited to, 2-aminoethanol or choline (e.g., -O-CH 2 -CH 2 -N + (CH 3 ) 3 ) group, etc., and is a glycerol derivative. Phosphatidylcholine is a phosphoglyceride in which choline is bonded to the glycerol moiety by a phosphodiester bond. Glycerol phosphoethanolamine is a 2-aminoethane group (e.g., -CH 2 -CH 2 -NH 3) is a phosphoglyceride bonded to the glycerol moiety by a phosphodiester bond. Amphiphilicity refers to, for example, a molecule or compound having both a hydrophobic moiety and a hydrophilic moiety under physiological conditions.

[0091] In the present specification, there is provided a formulation for CNS delivery of lipid-containing particles, such as lipid nanoparticles or microparticles. The lipid nanoparticles incorporate sphingolipid helper lipids, which assist in the delivery of the nanoparticles to cells, tissues, and organs of the central nervous system when delivered, for example, by intracerebroventricular injection into the cerebrospinal fluid of the spinal cord, brain, or brainstem, and / or into the brainstem, within the subarachnoid space, intracranially, or at the base of the skull. The sphingolipid provides excellent delivery to cells of the central nervous system when used as a helper lipid in the lipid particles described herein, as compared to previously described lipid nanoparticles.

[0092] A sphingolipid is a lipid containing a sphingoid base, any fatty acid, and any head group. The sphingoid base contains an unsaturated or saturated chain of carbon atoms (e.g., 18 carbon atoms) and has 1 to 3 hydroxyl groups and one amino group at the 2-position of the carbon chain. The general structure (excluding stereochemistry) of a sphingolipid is as follows:

Chemical formula

[0093] For example, the sphingolipid of formula (I) may have sphingosine as the sphingoid base, wherein R 3 is hydrogen, the carbon-carbon bond between a and b is a double bond, and the carbon-carbon bond between c and d is a single bond.

Chemical formula

[0094] The sphingolipid of formula (I) may have dihydrosphingosine as the sphingoid base, wherein R 3 is hydrogen, the carbon-carbon bond between a and b is a single bond, and the carbon-carbon bond between c and d is a single bond.

Chemical formula

[0095] The sphingolipid of formula (I) may have phytosphingosine as the sphingoid base, wherein R 3 is a hydroxyl group, the carbon-carbon bond between a and b is a single bond, and the carbon-carbon bond between c and d is a single bond.

Chemical formula

[0096] The sphingolipid of formula (I) may have dihydrophytosphingosine as the sphingoid base, wherein R 3 is a hydroxyl group, the carbon-carbon bond between a and b is a single bond, and the carbon-carbon bond between c and d is a double bond.

Chemical formula

[0097] R 1When it is a head group in formula (I), the head group may be bonded to an oxygen atom via a phosphodiester bond or a glycosidic bond. The head group may contain phosphocholine or phosphoethanolamine, and the phosphocholine or phosphoethanolamine may be bonded to the oxygen atom of formula (I) via a phosphodiester bond. Alternatively, the head group may contain a carbohydrate and may be bonded to an oxygen atom via a glycosidic bond. As used herein, "carbohydrate" includes one or more monosaccharides linked together by glycosidic bonds. The carbohydrate may contain monosaccharides. Alternatively, the carbohydrate may contain two monosaccharides (disaccharide), and the two monosaccharides are bonded via a glycosidic bond. Alternatively, the carbohydrate may contain 3 to 10 monosaccharides (oligosaccharide), or the carbohydrate may contain more than 10 monosaccharides (polysaccharide), and each monosaccharide is bonded to the next monosaccharide via a glycosidic bond. The carbohydrate may be a linear monosaccharide or a branched monosaccharide. The monosaccharide may optionally contain a sulfonate group, an amide group, a carboxylic acid group, a sialic acid group, or a combination thereof.

[0098] In formula (I), R 2 When it is a fatty acid chain, the acyl group of the fatty acid is bonded to the sphingoid base via an amide bond.

[0099] Non-limiting examples of suitable sphingolipids include ceramide, sphingomyelin, ceramide phosphoethanolamine, sphingosylphosphorylcholine, sphingosine, sphingoglycolipid, and combinations thereof.

[0100] The sphingolipid may be ceramide. Ceramide is composed of the general structure of formula (I), wherein R 1 is hydrogen and R 2 is a fatty acid bonded via an amide bond.

[0101] For example, the ceramide has the following general structure: [Chemical formula] may contain In the formula, R 3 may be hydrogen or a hydroxyl group. R 3 When R is hydrogen, the carbon-carbon bond between a and b may be a single bond or a double bond, and the carbon-carbon bond between c and d may be a single bond or a double bond. R 3 When R is a hydroxyl group, the carbon-carbon bond between a and b may be a single bond, and the carbon-carbon bond between c and d may be a single bond or a double bond. R 4 contains a saturated fatty acid chain, a monounsaturated fatty acid chain, a polyunsaturated fatty acid chain, an omega-hydroxy saturated fatty acid chain, an omega-hydroxy monounsaturated fatty acid chain, or an omega-hydroxy polyunsaturated fatty acid chain containing 1 to 31 carbon atoms, for example, 10 to 25 carbon atoms.

[0102] In the ceramide of formula (II), when R 4 contains a saturated fatty acid chain or an omega-hydroxy saturated fatty acid chain, the chain may be C n H 2n+1 where n may range from 1 to 31 or 10 to 25, including both ends. For example, the saturated fatty acid chain may be CH 3 C 3 H 7 C 5 H 11 C 7 H 15 C 9 H 19 C 11 H 23 C 13 H 27 C 15 H 31 C 17 H 35 C 19 H 39 C 21 H 43 C 23 H 47 C 25 H 51 C27 H 55 、 C 29 H 59 、 or C 31 H 63 may be.

[0103] In the ceramide of formula (II), when R 4 contains a monounsaturated fatty acid chain or an omega-hydroxy monounsaturated fatty acid chain, said chain is C n H 2n-1 and may be in the range of 10 - 25 or 17 - 23, including both ends. For example, said monounsaturated fatty acid chain is C 17 H 33 or C 23 H 45 may be.

[0104] For example, said ceramide may contain the following structure and is referred to as ceramide herein,

Chemical formula

[0105] Said sphingolipid may be sphingomyelin. Sphingomyelin is a phosphorus-containing sphingolipid and contains the general structure of formula (I), wherein R 1 is a phosphocholine head group bonded via a phosphodiester bond, and R 2 is a fatty acid bonded via an amide bond.

[0106] For example, said sphingomyelin has the following general structure:

Chemical formula

[0107] In the sphingomyelin of formula (III), R 5 When it contains a saturated fatty acid chain or an omega-hydroxy saturated fatty acid chain, the chain may be C n H 2n+1 and n may range from 1 to 31 or from 10 to 25, including both ends. For example, the saturated fatty acid chain may be CH 3 C 3 H 7 C 5 H 11 C 7 H 15 C 9 H 19 C 11 H 23 C 13 H 27 C 15 H 31 C 17 H 35 C 19 H 39 C 21 H 43 C 23 H 47 C 25 H 51 C 27 H 55 C 29 H 59 or C 31 H 63 may be.

[0108] In the sphingomyelin of formula (III), R 5 When it contains a monounsaturated fatty acid chain or an omega-hydroxy monounsaturated fatty acid chain, the chain is C n H 2n-1It may be, and n may be in the range of 10 to 25 or 17 to 23, including both ends. For example, the monounsaturated fatty acid chain is C 17 H 33 or C 23 H 45 and may be.

[0109] For example, the sphingomyelin has the following structure:

Chemical formula

[0110] The sphingolipid may be ceramide phosphoethanolamine. Ceramide phosphoethanolamine may include the general structure of formula (I), and R 1 is phosphoethanolamine, and R 2 is a fatty acid bonded via an amide bond.

[0111] For example, the ceramide phosphoethanolamine has the following general structure:

Chemical formula

[0112] In the ceramide phosphoethanolamine of formula (IV), R 6 when containing a saturated fatty acid chain or an omega-hydroxy saturated fatty acid chain, said chain may be C n H 2n+1 and n may range from 1 to 27 or from 10 to 25, including both ends. For example, said saturated fatty acid chain may be CH 3 、C 3 H 7 、C 5 H 11 、C 7 H 15 、C 9 H 19 、C 11 H 23 、C 13 H 27 、C 15 H 31 、C 16 H 33 、C 17 H 35 、C 19 H 39 、C 21 H 43 、C 22 H 45 、C 23 H 47 、C 25 H 51 、C 27 H 55 、C 29 H 59 、or C 31 H 63 and may be.

[0113] In the ceramide phosphoethanolamine of formula (IV), R 6 when containing a mono-unsaturated fatty acid chain or an omega-hydroxy mono-unsaturated fatty acid chain, said chain may be C n H 2n-1 and n may range from 10 to 25 or from 17 to 23, including both ends. For example, said unsaturated fatty acid chain may be C 17 H 33 or C 23 H 45 and may be.

[0114] The sphingolipid may be sphingosylphosphorylcholine. Sphingosylphosphorylcholine has the following structure:

Chem.

[0115] The sphingolipid may be sphingosine. Sphingosine has the following structure:

Chem.

[0116] The sphingolipid may be a glycosphingolipid. A glycosphingolipid is a sphingolipid containing a carbohydrate and includes the general structure of formula (I), where R 1 is a carbohydrate, and R 2 is a fatty acid bonded via an amide bond.

[0117] The glycosphingolipid may have the following general structure:

Chem.

[0118] The sphingoglycolipid has the following general structure:

Chemical formula

[0119] The sphingoglycolipid has the following general structure:

Chemical formula

[0120] In the glycosphingolipids of formulas (V) to (VII), R 7 , R 8 , or R 9 When containing a saturated fatty acid chain or an omega-hydroxy saturated fatty acid chain, said chain may be C n H 2n+1 , and n may range from 1 to 27 or 10 to 25, including both ends. For example, said saturated fatty acid chain may be CH 3 , C 3 H 7 , C 5 H 11 , C 7 H 15 , C 9 H 19 , C 11 H 23 , C 13 H 27 , C 15 H 31 , C 16 H 33 , C 17 H 35 , C 19 H 39 , C 21 H 43 , C 22 H 45 , C 23 H 47 , C 25 H 51 , C 27 H 55 , C 29 H 59 , or C 31 H 63 It may be.

[0121] In the glycosphingolipids of formulas (V) to (VII), R 7 , R 8 , or R 9 When containing a mono-unsaturated fatty acid chain or an omega-hydroxy mono-unsaturated fatty acid chain, said chain is Cn H 2n-1 may be, and n may be from 10 to 25 or from 17 to 23, including both ends. For example, the monounsaturated fatty acid chain is C 17 H 33 or C 23 H 45 and may be.

[0122] For example, the glucosylsphingolipid may have the following structure:

Chemical formula

[0123] For example, the galactosylsphingolipid may have the following structure:

Chemical formula

[0124] For example, the lactosylsphingolipid may have the following structure:

Chemical formula

[0125] For example, the sphingolipid may have the following general structure:

Chemical formula

[0126] In the sulfide of formula (VIII), R 10 When it contains a saturated fatty acid chain or an omega-hydroxy saturated fatty acid chain, said chain may be C n H 2n+1 and n may range from 1 to 27 or 10 to 25, including both ends. For example, said saturated fatty acid chain may be CH 3 C 3 H 7 C 5 H 11 C 7 H 15 C 9 H 19 C 11 H 23 C 13 H 27 C 15 H 31 C 16 H 33 C 17 H 35 C 19 H 39 C 21 H 43 C 22 H 45 C 23 H 47 C 25 H 51 C 27 H 55 C 29 H 59 or C 31 H 63 and may be.

[0127] In the sulfide of formula (VIII), R 10When it contains a mono-unsaturated fatty acid chain or an omega-hydroxy mono-unsaturated fatty acid chain, the chain may be C n H 2n-1 where n can range from 10 to 25 or from 17 to 23, including both ends. For example, the mono-unsaturated fatty acid chain may be C 17 H 33 or C 23 H 45 .

[0128] For example, the sulfatide may have the following structure:

Chem.

[0129] The sphingolipid may have the following general structure:

Chem.

[0130] The sphingolipid may have the following general structure:

Chem.

[0131] The sphingolipid may have the following general structure:

Chem.

[0132] The sphingolipid has the following general structure:

Chem.

Chemical formula

[0133] In the ganglioside of formula (IX), R 11 When it contains a saturated fatty acid chain or an omega-hydroxy saturated fatty acid chain, the chain may be C n H 2n+1 and n may range from 1 to 27 or 10 to 25, including both ends. For example, the saturated fatty acid chain is CH 3 C 3 H 7 C 5 H 11 C 7 H 15 C 9 H 19 C 11 H 23 C 13 H 27 C 15 H 31 C 16 H 33 C 17 H 35 C 19 H 39 C 21 H 43 C 22 H 45 C 23 H 47 C 25 H 51, C 27 H 55 , C 29 H 59 , or C 31 H 63 may be.

[0134] In the ganglioside of formula (IX), when R 11 contains a monounsaturated fatty acid chain or an omega-hydroxy monounsaturated fatty acid chain, said chain may be C n H 2n-1 , n may be 10 - 25 or 17 - 23, including both ends. For example, said monounsaturated fatty acid chain may be C 17 H 33 or C 23 H 45 may be.

[0135] For example, said ganglioside may have the following structure: [Chemical formula] may be a brain ganglioside containing

[0136] Said sphingolipid helper lipid is a sphingolipid that naturally exists in the brain and / or CNS tissues such as the CNS or brain tissues of vertebrates, mammals, or humans, and is referred to herein as "CNS sphingolipid" or "brain sphingolipid". Non-limiting examples of brain sphingolipids include sphingomyelin (brain SM), brain gangliosides, and brain sulfatides.

[0137] Lipid-containing particles such as lipid nanoparticles that provide excellent delivery to CNS cells are provided. Examples of lipid-containing particles described herein include, but are not limited to: helper sphingolipids; cholesterol or its derivatives; PEG-based compounds such as PEG-containing polymers or PEGylated fatty acid-containing compounds; and ionizable lipids (lipidoids).

[0138] Depending on their size, the lipid-containing particles may be described as lipid nanoparticles or lipid microparticles. The particles can be used to deliver any compatible cargo or active agent, such as, but not limited to, polynucleotides, drugs, proteins or peptides, small molecules, or gases. The particles can be used to deliver anionic or polyanionic cargo to cells, tissues, and / or organs of a patient's central nervous system. The anionic or polyanionic cargo may be a protein or peptide. The anionic or polyanionic cargo may be a nucleic acid, such as, but not limited to, mRNA, antisense reagents, RNAi agents, RNA, or DNA encoding genome-editing RNA, such as guide RNA (gRNA) or prime editing RNA (pegRNA), and mRNA encoding Cas9 or a Cas9 fusion protein, gene vectors or recombinant constructs, such as plasmids or other extrachromosomal or chromosomal targeting nucleic acids, recombinant or native viral genomes, DNA containing genes, ribozymes, or aptamers. For example, but not limited to, the agent or cargo may be RNA (such as mRNA, RNAi reagents, dsRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA (gRNA), long non-coding RNA (lncRNA), base editing gRNA (beRNA), prime editing gRNA (pegRNA), or transfer RNA (tRNA)). The cargo may be a therapeutic polypeptide or protein, or mRNA encoding an immunogen, such as mRNA that may be capped and optionally PEGylated, or may be non-coding.

[0139] The lipid particles described herein can also be incorporated into drug delivery devices, such as drugs, dosage forms, unit dosage forms, etc., and the lipid particles can be used to encapsulate agents containing polynucleotides, small molecules, proteins, peptides, metals, organometallic compounds, etc.

[0140] As is widely known, "nucleic acids" include naturally occurring DNA and RNA, as well as chemically modified nucleic acids. As a class, they may optionally include peptide nucleic acids (PNAs) having a neutral backbone, but modified peptide nucleic acids modified with anionic moieties such as gamma-modified PNA can also be utilized in the compositions and methods of the present invention. The nucleic acids useful in the compositions and methods described herein can be polyanionic nucleic acids that are overall negatively charged under neutral or physiological conditions, for example, in an aqueous solution at pH 6 to 8, such as water, blood, serum, Ringer's solution, or physiological saline. The nucleic acids may contain phosphorus-containing moieties such as phosphate and / or phosphorothioate moieties, and thus are polyanionic. Non-limiting examples of nucleic acids include, among others, RNAi agents, antisense reagents, aptamers, and ribozymes (see, for example, Bajan S, Hutvagner G. RNA-Based Therapeutics: From Antisense oligonucleotides to miRNAs. Cells. 2020 Jan 7;9(1):137; Invitrogen RNAi Handbook, ThermoFisher Scientific 2015; and Kilanowska, A., et al., In vivo and in vitro studies of antisense Oligonucleotides - a review, RSC Adv., 2020, 10, 34501). The nucleic acids may be unmodified (e.g., natural) or chemically modified (see, for example, Dar, S., et al., siRNAmod: A database of experimentally validated chemically modified siRNAs. Sci Rep 6, 20031(2016) and crdd.osdd.net / servers / sirnamod / ).

[0141] The diameter of the lipid-containing particles may range from 1 micrometer to 1,000 micrometers (micro particles). The diameter of the particles may range from 1 micrometer to 100 micrometers, from 1 micrometer to 10 micrometers, from 10 micrometers to 100 micrometers, from 100 micrometers to 1,000 micrometers, or from 1 to 5 micrometers. The diameter of the lipid particles may range from 1 nm to 1,000 nm (nano particles), from 1 nm to 100 nm, from 1 nm to 10 nm, from 10 nm to 100 nm, from 100 nm to 1,000 nm, from 20 nm to 2,000 nm, or from 1 to 5 nm. The diameter of the particles ranges from 1 pm to 1,000 pm, from 1 pm to 100 pm, from 1 pm to 10 pm, from 10 pm to 100 pm, from 100 pm to 1,000 pm, or from 1 to 5 pm.

[0142] The lipid particles can be prepared using any useful method. Among these, there are, among other methods, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, and simple and complex coacervation, but are not limited thereto.

[0143] The method for preparing the particles may be a double emulsion process and spray drying.

[0144] The conditions used in the preparation of the particles can be changed in order to obtain particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external form, "adhesiveness", shape, etc.). The method for preparing the particles and the conditions used (e.g., solvent, temperature, concentration, air flow rate, etc.) may also depend on the composition of the encapsulated drug and / or matrix.

[0145] Methods developed for making particles for delivering encapsulated agents are well described in the literature. In one example, the lipid-containing particles are prepared by microfluidics (see, e.g., Chen D, et al., Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc. 2012 Apr 25;134(16):6948-51 and Cayabyab, C, et al., “mRNA Lipid Nanoparticles: Robust low-volume production for screening high-value nanoparticle materials,” Document ID: mrnaspark-AN-1018, (2018) Precision NanoSystems, Inc., which describes a method for making lipid nanoparticles containing various components in suitable ratios). Briefly, an appropriate amount of the lipidoid, the sphingolipid helper lipid, the cholesterol or cholesterol derivative, and the PEG-based substance are mixed in a suitable solvent such as 90% ethanol and 10% 10 mM sodium citrate, and in 10 mM sodium citrate, an appropriate amount of cargo such as siRNA is mixed with, for example, but not limited to, a siRNA or mRNA to (lipidoid + cholesterol or cholesterol derivative + helper lipid + PEG-based substance) weight ratio of 1:2 to 1000, for example 1:4 to 1:50, for example, 1:10. For siRNA and mRNA, the lipidoid:siRNA ratio may range from 2:1 to 30:1, for example, for siRNA, the lipidoid:siRNA ratio may be 5:1, and for mRNA, the lipidoid:mRNA ratio may be 10:1. The amount of helper lipid in the lipid particles may range from 10 to 80 mol% of the total lipid, for example, the amount of lipidoid + cholesterol or cholesterol derivative + helper lipid + PEG-based substance in the lipid particles. The lipid particles can be formed by an automated device (such as a microfluidic device) or rapid pipetting operations.The particles may be diluted in a suitable aqueous solvent such as PBS and optionally dialyzed against the same or a different aqueous solvent.

[0146] If the particles prepared by any of the above methods have a size range outside the desired range, the particles can be size-specified, for example, using a sieve or a filter. The particles may also be coated. The particles may be coated with a targeting agent.

[0147] The lipid-containing particles include cholesterol or a derivative thereof, for example, 3β[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol). The lipid-containing particles include a PEG (poly(oxyethylene))-based substance, for example, a PEGylated fatty acid-containing compound or a PEG-containing block copolymer, such as a poloxamer. Non-limiting examples of PEG-based substances include PEG-ceramide, PEG-DMG, PEG-PE, poloxamer, or DSPE carboxy PEG. For example, in certain embodiments, the PEG-based substance is C14PEG2000DMG, C15PEG2000DMG, C16PEG2000DMG, C18PEG2000DMG, C14PEG2000ceramide, C15PEG2000ceramide, C16PEG2000ceramide, C18PEG2000ceramide, C14PEG2000PE, C15PEG2000PE, C16PEG2000PE, C18PEG2000PE, C14PEG350PE, C14PEG5000PE, poloxamer F-127, poloxamer F-68, poloxamer L-64, or DSPE carboxy PEG.

[0148] A lipidoid is a lipid-like molecule. An ionizable lipidoid is a lipidoid that forms ions under acidic or basic conditions. Non-limiting examples of ionizable lipidoids are generally provided in U.S. Patent No. 9,439,968, which forms lipidoids by conjugate addition of an alkyl acrylate to an amine. A general synthetic scheme for useful amino lipidoids prepared from an amine and an alkyl acrylate is shown in Figure 1A. For example, useful amines designated as 25, 32, 306, etc., and structures of alkyl acrylates such as O 10 O 11 O 12 O 13 O 14 are also provided. In the following examples, the lipidoid is designated with reference to the amine and alkyl acrylate used to make the ionizable lipidoid. For example, 306O 10 refers to N 1 -(3-aminopropyl)-N 1 -methylpropane-1,3-diamine conjugated with decyl acrylate, as shown in Figure 1B, and the technical name of 306O 10 is tetrakis(decyl) 3,3’,3’’,3’’’-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate.

[0149] Lipidoids for preparing LNPs for delivery to the CNS (including cells, tissues, organs, and / or organ systems) can include any combination, such as by Michael addition, of alkylamines having 1 to 5 amine moieties and alkyl or alkenyl acrylates having a pKa in the range of 3 to 7. The lipidoids for preparing LNPs for delivery to the CNS can include any combination permutation of the amines shown in FIGS. 2A-2C and one or more acrylates shown in FIGS. 3A-3C, such as those shown in FIGS. 1A and 1B. Lipidoids for preparing lipid-containing particles for mucosal delivery can include any combination permutation of the amines shown in FIGS. 2A-2C and one or more acrylates shown in FIGS. 3A-3C having a pKa in the range of 3 to 7, such as 5 to 7. Further examples of lipidoids are incorporated herein by reference for further exemplary lipidoid compounds and descriptions of their use, U.S. Patent Application Publication Nos. 20110256175A1 and 20200109113A1, and U.S. Patents Nos. 7939505B2, 8802863B2, 8969353B2, 9139554B2, and 9227917B2.

[0150] The lipidoid can be selected from lipidoids illustrated and described in connection with FIGS. 2A-3C such as Oi10 306 10 306O i10 503O 6,10 and 402O.

[0151] Methods and formulations, such as drugs, containing lipid nanoparticles for delivery to neurons and neuronal tissues, including cells, tissues, and organs of the CNS, such as, but not limited to, astrocytes, neurons, oligodendrocytes, neural stem cells, lymphocytes, and / or microglial cells, are also described herein. When used in patients, the lipid nanoparticles may rely on the ionizable lipid that is neutrally charged at physiological pH (i.e., 7.4). Once the lipid nanoparticles are taken up by cells, they are trapped in endosomes, which gradually become acidic and may degrade endosomal components. The ionizable lipid (lipidoid) is designed to ionize, i.e., become positively charged in the acidic endosome, causing endosomal membrane rupture and releasing the nucleic acid cargo into the cytoplasm to exert a therapeutic effect.

[0152] Intrathecal administration of a therapeutic agent, such as an API (active pharmaceutical ingredient) such as the nucleic acid in the LNP described herein, refers to injection into the cerebrospinal fluid of a patient, such as into the subarachnoid space of the brain or spinal cord, such as into the spine, and typically the lumbar spine (see, for example, Shah N, et al. Intrathecal Delivery System. [Updated 2022 Dec 9]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-). Examples of devices for intrathecal delivery include injection devices, intrathecal portals, pumps, and catheters, among other turnkey or generic devices. The LNP described herein can be provided as part of a kit that includes a syringe for injecting the LNP, or direct intrathecal injection using a pump or other device, such as, but not limited to, the Intrathecal Catheter System available from Harvard Apparatus, Holliston, Massachusetts, or other reservoirs or containers suitable for use in infusion, or a pump, such as the SynchroMedTM II Intrathecal Pump available from Medtronic, Minneapolis, Minnesota, particularly when repeated dosing is required, and an accompanying catheter. Brainstem injection can be performed similarly.

[0153] Intracerebroventricular (ICV) injection, including injection or infusion, i.e., delivery of an API via the ICV route, can be carried out using such standard techniques of the procedure and treatment. Intracerebroventricular (ICV), or intraventricular devices have been used in the treatment of various pediatric and adult central nervous system (CNS) disorders (see, e.g., Atkinson AJ Jr. Intracerebroventricular drug administration. Transl Clin Pharmacol. 2017 Sep;25(3):117-124.doi:10.12793 / tcp.2017.25.3.117.Epub 2017 Sep 15). The ICV portal, which may also be referred to as an Ommaya reservoir, can be used for delivery via the ICV route (see, e.g., Zubair A, De Jesus O. Ommaya Reservoir. [Updated 2023 Feb 12]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing;2023 Jan-).

[0154] The LNP composition (e.g., API) can be formulated or manufactured according to standard pharmaceutical practice and administered in a suitable carrier such as water, or water and / or buffer containing a suitable salt, such as those used for intrathecal or ICV delivery routes, including, for example but not limited to, water, saline such as physiological saline (e.g., 0.9% saline), buffered saline such as phosphate buffered saline (PBS), Ringer's solution, lactated Ringer's solution, or any other suitable carrier, and may also include any other suitable excipients or active ingredients, preservatives, rheology modifiers, antibiotics, analgesics, etc.

[0155] "Expression" or "gene expression" means the overall flow of information from a gene (which is not limited to, but includes a functional gene unit for producing a gene product in a cell, such as an RNA or protein, optionally modified after translation, or a functional / structural RNA, or another expression system encoded on a nucleic acid and containing other cis-acting elements such as a transcriptional promoter and response elements and / or enhancers; typically an expression sequence (open reading frame or ORF) encoding a protein or a functional / structural RNA, and a polyadenylation sequence) for producing a gene product (typically a protein, optionally modified after translation, or a functional / structural RNA). "Expression of a gene under the transcriptional control of a specified sequence", or "regulated by a specified sequence" means gene expression from a gene containing the specified sequence operably linked (typically cis, functionally bound) to the gene. The specified sequence can be all or part of the transcriptional elements (which are not limited to, but include promoters, enhancers and response elements) and may be capable of regulating and / or influencing the transcription of the gene either wholly or in part. A "gene for expression of the described gene product" is a gene capable of expressing the described gene product when placed in a suitable environment, i.e., when, for example, transformed, transfected, transduced into a cell, etc. and subjected to conditions suitable for expression. In the case of a constitutive promoter, "suitable conditions" means that it is typically only necessary to introduce the gene into a host cell. In the case of an inducible promoter, "suitable conditions" means the case where an amount of each inducer is administered to an expression system (e.g., a cell) effective to cause expression of the gene.

[0156] As used herein, the term "nucleic acid" refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acid analogs include, for example, but are not limited to, 2'-O-methyl-substituted RNA, locked nucleic acid, unlocked nucleic acid, triazole-linked DNA, peptide nucleic acid, morpholino oligomer, dideoxynucleotide oligomer, glycol nucleic acid, threose nucleic acid, and combinations thereof which may optionally contain ribonucleotide or deoxyribonucleotide residue(s). As used herein, in the context of nucleic acids and nucleic acid analogs, the term "nucleic acid" is used synonymously with "oligonucleotide", which is a short single-stranded structure composed of nucleotides. Oligonucleotides may be referred to by the descriptor "-mer" according to the length of the strand (i.e., the number of nucleotides). For example, a 22-nucleotide oligonucleotide is called a 22mer.

[0157] A "nucleic acid analog" is a composition comprising a nucleic acid base sequence disposed on a substrate such as a polymer backbone, and is capable of binding to DNA and / or RNA by Watson-Crick hybridization or Watson-Crick-like hydrogen-bond base pairing. Non-limiting examples of common nucleic acid analogs include peptide nucleic acid, such as γPNA, morpholino nucleic acid, phosphorothioate, locked nucleic acid (including 2'-O-4'-C-methylene bridge, its oxy, thio or amino forms), unlocked nucleic acid (wherein the C2'-C3' bond is cleaved), 2'-O-methyl-substituted RNA, threose nucleic acid, glycol nucleic acid, and the like.

[0158] Any form of gene editing can introduce or correct polymorphisms in tissues of the central nervous system, administered, for example, into the subarachnoid space, into the brain, or into the brainstem, according to the methods described herein. Single nucleotide polymorphisms or single nucleotide variants (SNPs or SNVs, respectively), or other disease-related mutations, polymorphisms, variants, etc. can be detected by sequencing, PCR, RT-PCR, or any useful method, and corrected using one or more editing tools such as the CRISPR and Cas9 system tools described herein. Similarly, dominant alleles, ancestral alleles, wild-type alleles, primary alleles, normal alleles (etc.) can be altered to change responses, interactions, regulatory pathways, etc. to treat disease, or to produce desired genotypes or phenotypes, for example, in animal breeding, to treat overreaction responses, or to treat hyperplasia or cancer. Thus, regardless of whether the reason and the associated genomic sequence are normal or abnormal, or whatever the degree of association between the allele and the disease risk, the relevant nucleotide sequence can be altered in a desired manner using gene editing.

[0159] As an example of a gene editing method, one or more nucleobases can be removed or edited using CRISPR-CAS9 editing (Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-CRISPR associated protein 9 (Cas9)) system, base editing, or prime editing. For example, sequences present in the genome of a subject or patient can be substituted, inserted, and / or deleted. Genes can be inactivated or modified using CRISPR-Cas9, or base editors or prime editors can be utilized. The CRISPR-Cas9 system, as well as base editors, include guide RNA (gRNA) or single guide RNA (sgRNA) and CRISPR associated protein 9 (Cas9) nuclease. Methods for identifying the DNA target strand and performing alterations in the target DNA (e.g., gene knockout in the target DNA strand, knock-in of a desired sequence, or base substitution) are within the capabilities of those skilled in the art.

[0160] The non-target DNA strand contains a specific protospacer adjacent motif (PAM) for the gRNA to bind to the target DNA strand. The PAM is a short nucleotide motif on the 3'-side with respect to the target site. In the case of the CRISPR-Cas9 system, the PAM is 5′-NGG-3′, where N is any nucleotide and G is guanine. The Cas9 nuclease cleaves 3 to 4 nucleotides upstream of the PAM sequence. The positions within the genome that different Cas proteins can target may be limited by the position of the PAM sequence and are known to those skilled in the art.

[0161] In CRISPR-Cas9 editing, when the Cas9 nuclease binds to the PAM and the gRNA binds to the target DNA strand, a double-strand break occurs in the gRNA sequence. Endogenous repair mechanisms such as non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or homologous recombination repair (HDR) are triggered by the double-strand break, resulting in gene knockout in the target DNA strand or, if a DNA template is present, knock-in at the desired sequence. The DNA template contains the desired sequence, which is flanked by sequences homologous to the regions upstream and downstream of the double-strand break.

[0162] The gRNA includes a CRISPR RNA (crRNA) that is a 17- to 20-nucleotide sequence complementary to the target DNA strand and a tracrRNA that functions as a binding scaffold for the Cas9 nuclease. The crRNA and the tracrRNA may exist as two separate RNA molecules. Alternatively, the sgRNA may include both the crRNA sequence and the tracrRNA sequence, and the crRNA sequence is fused to the scaffold tracrRNA sequence. The gRNAs of the base editing methods described below have canonical structures specific to each technique. One skilled in the art will select a gRNA or sgRNA that efficiently maximizes the on-target DNA cleavage while minimizing unintended off-target binding and cleavage effects (see Konstantakos et al. “CRISPR-Cas9 gRNA efficiency prediction: an overview of predictive tools and the role of deep learning. Nucleic Acids Res., 2022, 50(7):3616-3637 and “the Complete Guide to Understanding CRISPR sgRNA”, Synthego, 2023, www.synthego.com / guide / how-to-use-crispr / sgrna).

[0163] Alternatively, a base editing system can be used to convert G to another nucleobase. Base editing is a genome editing technology that uses a DNA base editor to directly generate precise point mutations without generating double-strand breaks. The DNA base editor can include a fusion between catalytically dead Cas9 (dCas9) or nickase Cas9 (nCas9) fused to a single-stranded DNA (ssDNA)-specific deaminase and a single-guide RNA (sgRNA). The d / nCas9 recognizes a specific sequence named protospacer adjacent motif (PAM), and the DNA is unwound due to the complementarity between the sgRNA and the DNA sequence that is usually located upstream of the PAM (“protospacer”). Next, the opposite DNA strand becomes accessible to the deaminase that converts the base located in a specific stretch of DNA of the protospacer (see, for example, Antoniou P, et al. Base and Prime Editing Technologies for Blood Disorders. Front Genome Ed. 2021 Jan 28;3:618406). When the DNA base editor binds to the target DNA strand, a substitution of a small segment of ssDNA occurs as an “R-loop” as a result of base pairing between the sgRNA and the target DNA strand. Thus, the DNA base within the ssDNA is a substrate for deamination and is subsequently modified by the deaminase enzyme.The DNA base editor can be a cytosine base editor (CBE) that converts a C / G base pair into a T / A base pair or an adenine base editor (ABE) that converts an A / T base pair into a G / C base pair (see, for example, Qi et al. “Base Editing Mediated Generation of Point Mutations Into Human Pluripotent Stem Cells for Modeling Disease”, Frontiers in Cell and Developmental Biology, 2020, 8(590581):1-12; Nishida K, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science. 2016 Sep 16;353(6305):aaf8729; Komor AC, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016 May 19;533(7603):420-4; and Gaudelli NM, et al. Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage. Nature. 2017 Nov 23;551(7681):464-471).

[0164] Another method of editing SNVs is prime editing, which is disclosed in U.S. Patent No. 11,447,770B1, which is incorporated herein by reference in its technical disclosure, and in related publications (see also International Patent Publication No. 2020191242A1 and Anzalone AV, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019 Dec;576(7785):149-157). Prime editors (PEs) containing a complete description of the pegRNA are presented in these documents, and methods of in vivo delivery of prime editor materials, such as viral vectors such as AAV particles encoding the prime editor, are described in the said patent publication and related publications.

[0165] Prime editing is a "search and replace" gene editing method that fuses Moloney murine leukemia virus reverse transcriptase (M-MLV RT) to the C-terminus of the Cas9 H840A nickase. The fusion enzyme is directed to targeted insertions, deletions, and any possible base-base conversions using a prime editing guide RNA (pegRNA). The pegRNA directs the nickase to the target site by homology to the genomic DNA locus. Longer pegRNAs also encode the desired editing on the primer binding site (PBS) and RT template. Prime editing has gone through several versions. In PE1, the pegRNA directs the Cas9 nickase towards the target sequence, where a nick is introduced into the non-target strand, generating a 3' flap. The 3' flap binds to the primer binding site (PBS) of the pegRNA, and the desired edit is incorporated into the DNA by reverse transcription. The edited DNA strand replaces the unedited 5' flap, and the resulting heteroduplex is degraded by the cell's mismatch repair (MMR) system. Alternatively, the edited 3' flap can be excised, leaving the target sequence unchanged but available as a substrate for another round of prime editing.

[0166] In the PE2 system, mutations were introduced into the RT enzyme to increase its activity, enhance the binding between the template and PBS, increase the processing ability, and improve the thermal stability. In addition to the PE2 Cas9 nickase-penta mutant RT fusion enzyme and pegRNA, PE3 uses an additional simple sgRNA, which causes a break in the unedited strand at a site near the Cas9 nickase. The newly edited strand is preferred as the template for repair during heteroduplex cleavage. However, the process of double nicking slightly increases indel formation. By designing the sgRNA using a spacer that binds only to the edited strand as in the PE3b system, nicking of the unedited strand is induced only after the editing has occurred. PE4 and PE5 are also described (Chen PJ, et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell. 2021 Oct 28;184(22):5635-5652.e29). Plasmids useful for performing prime editing are commercially available from Addgene (www.addgene.org / crispr / prime-edit / ). "Prime editing" includes all versions of the prime editing method, including but not limited to the PE1, PE2, PE3, PE3b, PE4, and PE5 versions. pegRNAs include, but are not limited to, their variations for use in many of the variations of prime editing such as epegRNA (Nelson JW, et al. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol. 2022 Mar;40(3):402-410).

[0167] Computer-based tools have been developed for the automated generation of pegRNAs (see, for example, Morris et al. Automated design of CRISPR prime editors for 56,000 human pathogenic variants. iScience. 2021 Oct 30;24(11):103380, the Prime Editing Design Tool, a useful tool for identifying pegRNAs, is available at primeedit.nygenome.org / ; Hwang GH, et al. PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing. Nucleic Acids Res. 2021 Jul 2;49(W1):W499-W504; Hsu JY, et al. PrimeDesign software for rapid and simplified design of prime editing guide RNAs. Nat Commun. 2021 Feb 15;12(1):1034; and Chow RD, et al. A web tool for the design of prime-editing guide RNAs. Nat Biomed Eng. 2021 Feb;5(2):190-194). A pegRNA can include a protospacer sequence for recognizing the target sequence, a reverse transcriptase template (RTT) including the desired edit, and a primer binding site (PBS) for activation of the reverse transcriptase. As described above, several PEs have been developed and different gRNAs can be used depending on the type of prime editor (e.g., PE2 vs. PE3). For example, in contrast to PE2 which only requires a pegRNA, PE3 also requires a nicking guide RNA (ngRNA) to increase the prime editing efficiency (Hwang GH, et al. Nucleic Acids Res. 2021 Jul 2;49(W1):W499-W504).Although pegRNAs and ngRNAs, when applicable, can be developed by those skilled in the art without using a computer, computational tools such as those described above can be used for prime editing and for effectively designing pegRNAs and other useful reagents.

[0168] The CRISPR / Cas9, base editing, and prime editing, the necessary components, such as nucleic acids (including their mixtures of RNA, DNA, or nucleic acid analogs) encoding gRNA, template, pegRNA, ngRNA, Cas9, Cas9 nickase, or Cas9 fusion protein, etc., can be delivered by any effective means, but in the context of the present disclosure, they are delivered by LNPs. The introduced substances can take any useful form, but a DNA plasmid or recombinant viral genome containing sequences for the expression of the necessary reagents; and / or mRNA for the translation of the reagents may be included, together with suitable guide RNAs, such as gRNA or pegRNA, and other useful nucleic acid reagents such as ngRNA. DNA or RNA useful for the implementation of gene editing methods such as CRISPR / Cas9 editing methods including single-base editing such as CBE, ABE, or prime editing can be delivered to the CNS in accordance with and / or compliant with the methods and reagents described herein.

[0169] Gene editing tools have been delivered by LNPs, but the present disclosure enhances delivery to the CNS by use of sphingolipid-modified LNPs. In one example, Rosenblum et al. (Rosenblum D, et al. CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy. Sci Adv. 2020 Nov 18;6(47):eabc9450) delivered a gene editing tool intracranially against PLK1 to treat invasive glioblastoma multiforme and obtained good results. Rosenblum et al. also describe antibody targeting of LNPs. See, among others, Onuma H et al. (Onuma H, et al. Lipid nanoparticle-based ribonucleoprotein delivery for in vivo genome editing. J Control Release. 2023 Mar;355:406-416) and Li B et al. (Li B, et al. Combinatorial design of nanoparticles for pulmonary mRNA delivery and genome editing. Nat Biotechnol. 2023 Mar 30). Accordingly, the LNPs described herein are expected to be able to deliver therapeutic gene editing nucleic acids for effective gene editing.

[0170] LNPs have been used to deliver DNA. LNPs are generally considered RNA delivery compositions. That being said, DNA can effectively deliver, for example, but not limited to, plasmid DNA, linear DNA, and viral genomes such as adenovirus (Ad) or adeno-associated virus (AAV) genomes. The LNPs described herein can be used to deliver DNA. The ratio of the components of the sphingolipid-containing LNPs may need to be manipulated to optimize delivery, which is within the ability of those skilled in the art. For example, among others, see Zhu Y et al. (Multi-step screening of DNA / lipid nanoparticles and co-delivery with siRNA to enhance and prolong gene expression. Nat Commun. 2022 Jul 25;13(1):4282), Algarni et al. (In vivo delivery of plasmid DNA by lipid nanoparticles: the influence of ionizable cationic lipids on organ-selective gene expression. Biomater Sci. 2022 May 31;10(11):2940-2952), and Scalzo et al. (Ionizable Lipid Nanoparticle-Mediated Delivery of Plasmid DNA in Cardiomyocytes. Int J Nanomedicine. 2022 Jun 30;17:2865-2881). Therefore, the LNPs described herein are expected to be able to deliver therapeutic DNA for the effective production of gene products.

[0171] Messenger RNA has been successfully delivered to the brain. For example, ischemic stroke has been shown to be treatable by delivery of heme oxygenase 1 (HO1) mRNA by self-replicating mRNA (Rep-mRNA) developed using a replicon system from Venezuelan equine encephalitis virus (Kim M, et al. Delivery of self-replicating messenger RNA into the brain for the treatment of ischemic stroke. J Control Release. 2022 Oct;350:471-485. For an example of the sequence of deliverable HO1 mRNA, also see NCBI Reference Sequence: NM_002133.3). Intracerebroventricular delivery of synthetically modified tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA using TransIT®-mRNA and jetPEI® showed efficacy in a mouse glioblastoma model (Peng H, et al. Intracranial delivery of synthetic mRNA to suppress glioblastoma. Mol Ther Oncolytics. 2021 Dec 14;24:160-170). Finally, intracerebroventricular delivery of mRNA encoding brain-derived neurotrophic factor (BDNF) using polyplex nanomicelles, a polymeric carrier, was shown to increase the survival rate of hippocampal neurons after transient global ischemia (Fukushima Y, et al. Treatment of ischemic neuronal death by introducing brain-derived neurotrophic factor mRNA using polyplex nanomicelle. Biomaterials. 2021 Mar;270:120681). The TRAIL and BDNF mRNA sequences are widely known and available.The production of mRNA for use in the sphingolipid LNPs described herein can be achieved by isolating the mRNA according to well-established methods using any suitable gene expression system (see, for example, Rosa SS, et al. mRNA vaccines manufacturing: Challenges and bottlenecks. Vaccine. 2021 Apr 15;39(16):2190-2200). Many companies, among them ThermoFisher Scientific, offer mRNA large-scale production services. Thus, the LNPs described herein are expected to be able to deliver therapeutic RNA for the effective treatment of diseases such as ischemia, stroke, ischemia / reperfusion injury, neuroinflammation, neurodegenerative diseases, monogenic neuropathies, and cancer, such as, but not limited to, glioblastoma.

Example

[0172] Example 1 Helper lipid screening For mRNA delivery to mouse and human microglial cells, various helper lipids were incorporated into lipid nanoparticles.

[0173] Materials and methods The mRNA was N(1)-methylpseudouridine (m1ψ)-modified mRNA encoding green fluorescent protein (GFPmRNA).

[0174] Lipid nanoparticles (LNPs) were formulated with 16 mol% helper lipid, 35 mol% ionizable lipidoid 306 (as a proof-of-concept example) Oi10 , 46.5 mol% cholesterol and 2.5 mol% 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C 14 -PEG 2000) was incorporated. The helper lipids were 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (PG), 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (PA), 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), glycero-ethylphosphocholine EPC, brain sphingomyelin (brain SM), brain ganglioside extract, brain sulfatide, N-oleoyl-D-erythro-sphingosine (referred to as ceramide in the examples), C18 glucosylsphingoglycolipid (referred to as glucosylceramide in the examples), C18 lactosylsphingoglycolipid (referred to as lactosylceramide in the examples), or C18 galactosylsphingoglycolipid (referred to as galactosylceramide in the examples).

[0175] Lipidoid, helper lipid, cholesterol, and C 14 -PEG 2000 was dissolved in reagent-grade ethanol at 1 to 10 milligrams per milliliter (mg / mL). GFP mRNA was dissolved in 10 mM sodium citrate. The citrate buffer was added to the lipid solution at a volume ratio of 1:10. The resulting lipid solution was added to an equal volume of the mRNA solution at a lipidoid:mRNA mass ratio of 10:1 and then mixed well. Finally, an equal volume of phosphate-buffered saline (PBS) was added to the ethanol-citrate mixture and this was mixed well. Lipid nanoparticles for in vitro and in vivo experiments were formulated at final mRNA concentrations of 5 μg / mL and 90 μg / mL, respectively. The lipid nanoparticles used in the in vivo experiment were dialyzed against 2 liters (L) of PBS in a 3 kilodalton (kDa) molecular weight cut-off dialysis cassette for 90 minutes.

[0176] Cell culture - Human microglial cells (HCM3) Human microglial cells (HCM3) were cultured in EMEM / 10% FBS / 1% Pen / Strep / 0.1% Fungizone at 37 °C / 5% CO 2 and split with trypsin before reaching confluence. After the cells were allowed to adhere or settle for 24 hours, LNP administration was performed. 20 microliters of LNP per well was added to the cell culture medium at an initial concentration of 5 μg / mL GFP mRNA and incubated for 110 hours. HCM3 control cells were treated with PBS or naked GFP mRNA. The transfection efficiency was measured in real time every 2 hours using an IncuCyte ZOOM Live-Cell imaging system.

[0177] Cell culture - Mouse microglial cells (SIM-A9) Mouse microglial SIM-A9 cells were cultured in DMEM-F12 / 10% FBS / 5% horse serum / 1% Pen / Strep / 0.1% Fungizone at 37 °C / 5% CO 2 and split with trypsin before reaching confluence. After the cells were allowed to adhere or settle for 24 hours, LNP administration was performed. A physiologically appropriate mucin layer was created by adding 25 μL of 5% mucin diluted in medium covered with an additional 155 μL of medium. 20 microliters of LNP per well at an initial concentration of 5 μg / mL GFP mRNA was added to the cell culture medium and then incubated for 110 hours. SIM-A9 control cells were treated with PBS or naked GFP mRNA. The transfection efficiency was measured in real time every 2 hours using an IncuCyte ZOOM Live-Cell imaging system.

[0178] Cell culture - Human glioblastoma cells (U87MG) Human glioblastoma U87MG cells were cultured in DMEM / 10% FBS / 1% Pen / Strep / 0.1% Fungizone at 37 °C / 5% CO 2Cultured and split with trypsin before reaching confluence. After the cells were allowed to adhere or sediment for 24 hours, LNP administration was performed. A physiologically appropriate mucin layer was created by adding 25 μL of 5% mucin diluted in the medium covered with 155 μL of medium. 20 microliters of LNP at an initial concentration of 5 μg / mL GFP mRNA per well was added to the cell culture medium and then incubated for 110 hours. U87MG control cells were treated with PBS or naked GFP mRNA. The transfection efficiency was measured in real time every 2 hours using an IncuCyte ZOOM Live-Cell imaging system.

[0179] Cell culture - mouse glioblastoma cells (GL261) Mouse glioblastoma GL261 cells were cultured in DMEM / 10% FBS / 1% Pen / Strep / 0.1% Fungizone at 37 °C / 5% CO 2 Cultured and split with trypsin before reaching confluence. After the cells were allowed to adhere or sediment for 24 hours, LNP administration was performed. A physiologically appropriate mucin layer was created by adding 25 μL of 5% mucin diluted in the medium covered with 155 μL of medium. 20 microliters of LNP at an initial concentration of 5 μg / mL GFP mRNA per well was added to the cell culture medium and then incubated for 110 hours. GL261 control cells were treated with PBS or naked GFP mRNA. The transfection efficiency was measured in real time every 2 hours using an IncuCyte ZOOM Live-Cell imaging system.

[0180] Results and discussion The total green GFP fluorescence intensity taken up by HCM3, SIM-A9, U87MG, and GL261 cells when exposed to 13 different GFP mRNA-LNPs (n = 4) is shown in Figures 4A - 4D. The cell confluence (%) of HCM3, SIM-A9, U87MG, and GL261 cells when exposed to 13 different GFP mRNA-LNPs (n = 4) is shown in Figures 5A - 5D.

[0181] Lipid nanoparticles formulated with sphingolipid helper lipids (e.g., brain sphingomyelin, brain ganglioside extract, brain sulfatide, ceramide, lactosylceramide, and galactosylceramide) resulted in high transfection efficiency compared to LNP formulations prepared using other helper lipids.

[0182] Example 2 In Vivo Intracerebroventricular LNP Administration

[0183] Materials and Methods LNP was formulated with 16 mol% DOPE, 35 mol% ionizable lipid iodide (306 as a proof-of-concept example Oi10 ), 46.5 mol% cholesterol and 2.5 mol% C 14 -PEG 2000 . The mRNA was either luciferase mRNA (mLuc) or a combination of mLuc and mCherry mRNA (mCherry). LNP was prepared according to the method described in Example 1. The LNP was formulated to contain 0.5 mg / kg of mRNA.

[0184] All in vivo experiments were performed using male C57BL / 6NCrl (Charles River) mice at least 6 weeks old. Mice were independently injected intracerebroventricularly with (1) LNP containing mLuc, or (2) LNP containing a combination of mLuc and mCherry. One hour, 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, or 48 hours after injection, 130 μL of 30 mg / mL D-luciferin was injected intraperitoneally into mice injected with LNP containing mLuc. Six hours after injection, 130 μL of 30 mg / mL D-luciferin was injected intraperitoneally into mice injected with LNP containing mLuc and mCherry. Fifteen minutes after luciferin administration, the mice were anesthetized with CO 2Euthanasia was performed by asphyxiation and atlantoaxial dislocation. Organs were removed, excess blood was blotted away, and the organs were placed on black drawing paper. Luminescence signals were measured using an In Vivo Imaging System (Perkin Elmer), and luminescence images were juxtaposed with bright-field images. Total luminescence flux (p / s) for each organ was calculated using Living Image (registered trademark) software. Organs used for histological examination were immediately placed in 10% neutral buffered formalin, stored at 4 °C for 4 days, washed in PBS, and stored in 70% ethanol at 4 °C.

[0185] Results and Discussion Results of in vivo luciferase expression kinetics are shown in FIGS. 6A and 6B. It was found that 3 hours or 6 hours is the optimal time to evaluate luciferase expression in the mouse brain. To evaluate overall mRNA transfection, the brain was divided into three regions (forebrain / midbrain / hindbrain and spinal cord), and it was confirmed that delivery across the entire mouse brain was enabled via the intrathecal route (see FIGS. 7A - 7B).

[0186] Multiplex mRNA delivery of LNP containing mLuc and mCherry was analyzed 6 hours after injection. The data obtained are shown in FIGS. 8A - 8C, which show successful transfection with luciferase and mCherry mRNA. Thus, multiple mRNAs were delivered to the brain by intrathecal injection of LNP.

[0187] Example 3 Evaluation of Helper Lipids by Intrathecal LNP Administration in Vivo

[0188] Materials and Methods Independently for LNP, (1) 16 mol% DOPE, 35 mol% ionizable lipidoid (306 as a proof - of - concept example Oi10 ), 46.5 mol% cholesterol, and 2.5 mol% C 14 -PEG 2000 ; (2) 16 mol% DOPS, 35 mol% ionizable lipidoid (306 as a proof - of - concept example Oi10) 46.5 mol% cholesterol and 2.5 mol% C 14 -PEG 2000 ; or (3) 16 mol% DOTAP, 35 mol% ionizable lipidoid (306 as a proof-of-concept example) Oi10 ) 46.5 mol% cholesterol and 2.5 mol% C 14 -PEG 2000 were formulated. The mRNA was a combination of mLuc, Cre recombinase mRNA (mCre), or mRNA of Cas9 and sgRNA of mouse CD81 (Synthego Corporation). The scrambled guide control was obtained from Synthego Corporation. The LNPs were prepared according to the method described in Example 1. The LNPs containing mLuc or mCre were formulated to contain 0.5 mg / kg of mRNA. The LNPs containing a combination of mRNA of Cas9 and sgRNA of mouse CD81 were formulated to contain 0.5 mg / kg of mRNA of Cas9 and 0.05 mg / kg of sgRNA of mouse CD81, and the ratio of cas9 mRNA to sgRNA was 4.7:1 (w / w). The formulations containing the scrambled guide control were the same as the CD81 on-target formulations except that the guide was scrambled.

[0189] Evaluation of mRNA delivery to the brain Male C57BL / 6J (Charles River) mice or male Ai9 reporter mice (Jackson Laboratory) at least 6 weeks old were used in the in vivo experiments. Independently in C57BL / 6NCrl mice, (1) DOPE helper lipid LNP containing mLuc; (2) DOPS helper lipid LNP containing mLuc; or (3) DOTAP helper lipid LNP containing mLuc were injected into the subarachnoid space. Independently in Ai9 mice, (1) DOPE helper lipid LNP containing mCre; (2) DOPS helper lipid LNP containing mCre; or (4) DOTAP helper lipid LNP containing mCre were injected into the subarachnoid space. Six hours after injection, 130 μL of 30 mg / mL D-luciferin was intraperitoneally injected into C57BL / 6NCrl mice. Ninety-six hours after injection, 130 μL of 30 mg / mL D-luciferin was intraperitoneally injected into Ai9 mice. The mice were euthanized and the organs were prepared and analyzed as described in Example 2.

[0190] Cas9-Mediated Gene Knockout in Mice Independently in male C57BL / 6 mice, (1) DOPE helper lipid LNP containing Cas9 mRNA and sgRNA of mouse CD81; (2) DOPS helper lipid LNP containing Cas9 mRNA and sgRNA of mouse CD81; or (3) DOTAP helper lipid LNP containing Cas9 mRNA and sgRNA of mouse CD81 were injected into the subarachnoid space. One week after injection, 130 μL of 30 mg / mL D-luciferin was intraperitoneally injected into C57BL / 6NCrl mice. The mice were euthanized and the organs were prepared and analyzed as described in Example 2. The brain was collected from the mice, mechanically digested into a single cell suspension, and analyzed using flow cytometry to evaluate CD81 knockout. To evaluate the success of Cas9 expression, another group of animals was euthanized 24 hours after LNP administration.

[0191] Results and Discussion

[0192] mRNA Delivery to the Brain As shown in FIGS. 9A-9B, all LNPs were transported to the brain regardless of the charge of the helper lipid, and DOTAP resulted in the highest brain luciferase expression. The LNPs administered to Ai9 mice together with mCre expressed tdTomato upon Cre-mediated recombination. This revealed which cells translate mCre rather than the amount of protein expressed (FIGS. 10A-10I). 85-95% of microglia (CD11b+), 35-75% of astrocytes (GFAP+), 35-75% of neurons (NeuN+), 35-85% of neural stem cells (CD133+), 60-95% of oligodendrocytes, and 15-60% of lymphocytes (CD45+CD3+) translated mCre. Thus, intrathecal injection of mRNA in LNPs successfully transfected brain cells in vivo.

[0193] Cas9-mediated gene knockout in mice As shown in FIGS. 11A-11L, CD81 was effectively knocked out in microglial cells, neurons, and oligodendrocytes.

[0194] Example 4 Intrathecal LNP administration in vivo

[0195] Materials and methods Independently for LNPs, (1) 16 mol% ceramide, 35 mol% ionizable lipidoid (306 as a proof-of-concept example Oi10 ), 46.5 mol% cholesterol, and 2.5 mol% C 14 -PEG 2000 ; (2) 16 mol% glucosylceramide, 35 mol% ionizable lipidoid (306 as a proof-of-concept example Oi10 ), 46.5 mol% cholesterol and 2.5 mol% C 14- PEG 2000 ; (3) 16 mol% galactosylceramide, 35 mol% ionizable lipidoid (306 as a proof-of-concept example Oi10 ), 46.5 mol% cholesterol and 2.5 mol% C 14 -PEG 2000; or (4) 16 mol% lactosylceramide, 35 mol% ionizable lipidoid (306 as a proof-of-concept example) Oi10 ), 46.5 mol% cholesterol and 2.5 mol% C 14 -PEG 2000 were formulated. The mRNA was mLuc, mCre, a combination of mLuc and mCherry, or a combination of Cas9 mRNA and mouse CD81 sgRNA. The LNP was prepared according to the method described in Example 1. The LNP was formulated to contain 0.5 mg / kg mRNA. The LNP containing a combination of Cas9 mRNA and mouse CD81 sgRNA was formulated to contain 0.5 mg / kg of Cas9 mRNA and 0.05 mg / kg of mouse CD81 sgRNA, and the ratio of cas9 mRNA to sgRNA was 4.7:1 (w / w).

[0196] Evaluation of mRNA delivery to the brain Male C57BL / 6 (Charles River) mice or male and female Ai9 reporter mice (Jackson Laboratory) at least 6 weeks old were used in the in vivo experiments. Independently in C57BL / 6NCrl mice, (1) ceramide helper lipid LNP containing mLuc; (2) glucosylceramide helper lipid LNP containing mLuc; (3) galactosylceramide helper lipid LNP containing mLuc; (4) lactosylceramide helper lipid LNP containing mLuc; (5) ceramide helper lipid LNP containing a combination of mLuc and mCherry; (6) glucosylceramide helper lipid LNP containing a combination of mLuc and mCherry; (7) galactosylceramide helper lipid LNP containing a combination of mLuc and mCherry; or (8) lactosylceramide helper lipid LNP containing a combination of mLuc and mCherry were injected into the subarachnoid space. Independently in male or female Ai9 mice, (1) ceramide helper lipid LNP containing mCre; (2) glucosylceramide helper lipid LNP containing mCre; (3) galactosylceramide helper lipid LNP containing mCre; or (4) lactosylceramide helper lipid LNP containing mCre were injected into the subarachnoid space. One hour, 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, or 48 hours after injection, 130 μL of 30 mg / mL D-luciferin was intraperitoneally injected into the mice injected with LNP containing mLuc. At 6 hours after injection, 130 μL of 30 mg / mL D-luciferin was intraperitoneally injected into the mice injected with LNP containing mLuc and mCherry. At 96 hours after injection, 130 μL of 30 mg / mL D-luciferin was intraperitoneally injected into the mice injected with LNP containing mCre. The mice were euthanized and the organs were prepared and analyzed as described in Example 2. Single cell suspensions were collected from the brains of mice treated with LNP containing mCre and prepared for flow cytometry analysis. Also, the brains were fixed, embedded, stained for central nervous system cells, and imaged for tdTomato fluorescence using a confocal microscope.

[0197] Cas9-Mediated Gene Knockout in Mice Independently, male C57BL / 6NCrl mice were injected intracisternally with (1) a ceramide helper lipid LNP containing Cas9 mRNA and a mouse CD81 sgRNA; (2) a galactosylceramide helper lipid LNP containing Cas9 mRNA and a mouse CD81 sgRNA; or (3) a lactosylceramide helper lipid LNP containing Cas9 mRNA and a mouse CD81 sgRNA. One week after injection, 130 μL of 30 mg / mL D-luciferin was injected intraperitoneally into the C57BL / 6NCrl mice. The mice were euthanized and the organs were prepared and analyzed as described in Example 2. Brains were harvested from the mice, mechanically digested into single cell suspensions, and analyzed using flow cytometry to evaluate CD81 knockout. To evaluate the success of Cas9 expression, another group of animals was euthanized 24 hours after LNP administration.

[0198] Toxicity evaluation Independently, male and female C57BL / 6NCrl mice were injected intracisternally with (1) a ceramide helper lipid LNP containing mLuc; (2) a glucosylceramide helper lipid LNP containing mLuc; (3) a galactosylceramide helper lipid LNP containing mLuc; (4) a lactosylceramide helper lipid LNP containing MLuc; or (5) PBS. Blood was collected from the mice 1 hour, 3 hours, 6 hours, 24 hours, or 48 hours after injection. Serum was separated and evaluated for inflammatory cytokines using enzyme-linked immunosorbent assay (ELISA).

[0199] Results and discussion

[0200] mRNA delivery to the brain The multiplexed mRNA delivery of LNPs containing mLuc and mCherry was analyzed 6 hours after injection. The data obtained are shown in FIGS. 12A-12B, from which it can be seen that mRNA transfection was successful for luciferase and mCherry. The data obtained from LNPs prepared using ceramide, glucosylceramide, lactosylceramide, and galactosylceramide were compared with LNPs prepared using DOPE, DOPS, and DOPAP. Thus, multiple mRNAs were delivered to the brain by intracerebroventricular injection.

[0201] All lipid LNPs formulated with ceramide, glucosylceramide, galactosylceramide, or lactosylceramide successfully transfected the mouse brain with mCre after a single intracerebroventricular injection (FIGS. 13A-13J). The highest transfection effect among the tested LNPs was obtained with LNPs formulated with ceramide and lactosylceramide. All LNPs were able to transfect approximately 80% of neurons in the brain, as confirmed using immunostaining for different central nervous system (CNS) cell types (see FIG. 14). Overall, the data indicate that intracerebroventricular injection of mRNA in LNPs successfully transfected neurons and other brain cells in vivo. Cas9-Mediated Gene Knockout in Mice

[0202] As shown in FIGS. 15A-15L, most of the CD81 gene editing occurred in neurons compared to other cell types. In addition, CD81 gene editing in the mouse brain was confirmed by gene sequencing, as presented in FIGS. 16A-16H. The multiple bands in the CD81 sgRNA LNP in FIG. 16A indicate Cas9-mediated gene cleavage. Indels were present at the CD81 locus corresponding to Cas9-mediated editing.

[0203] Toxicity Assessment From the data, it can be seen that some inflammatory cytokines increased by 1-2 times within 3-6 hours after LNP injection, but this disappeared within 24 hours of injection, and as a result, no severe inflammatory reaction occurred. However, a transient increase in inflammatory cytokines was also observed in the PBS control group, suggesting that the administration route rather than the LNP formulation caused the inflammatory reaction.

[0204] Therefore, intrathecal administration of LNP formulated with ceramide, glucosylceramide, galactosylceramide, or lactosylceramide was well tolerated in mice.

[0205] The present invention has been described with respect to certain exemplary embodiments, dispersible compositions, and their use. However, those skilled in the art will understand that various substitutions, modifications, or combinations of any of the exemplary embodiments can be made without departing from the spirit and scope of the present invention in the art. Therefore, the present invention is not limited to the description of the exemplary embodiments, but is limited by the scope of the appended claims as originally filed.

Claims

**Claim 1** A method for delivering a therapeutic agent to a tissue of a patient's central nervous system (CNS), comprising administering to the tissue of the patient's CNS a composition comprising lipid-containing particles comprising a therapeutic agent, wherein the lipid-containing particles are: a sphingolipid helper lipid; cholesterol or a derivative thereof; a PEG-based compound such as a PEG-containing polymer or a PEGylated fatty acid-containing compound; and an ionizable lipidoid The method further comprising. **Claim 2** The method according to claim 1, wherein the lipid-containing particles are lipid nanoparticles. **Claim 3** The method according to claim 1 or 2, wherein the lipid-containing particles are administered into the subarachnoid space, into the brain, or into the brainstem of the patient. **Claim 4** The method according to any one of claims 1 to 3, wherein the sphingolipid is ceramide, sphingomyelin, ceramide phosphoethanolamine, sphingosylphosphorylcholine, sphingosine, sphingoglycolipid, or any combination of two or more thereof. **Claim 5** The method according to claim 4, wherein the sphingolipid is ceramide, glucosylceramide, galactosylceramide, or lactosylceramide. **Claim 6** The method according to any one of claims 1 to 5, wherein the sphingolipid is a CNS sphingolipid, a brain sphingolipid, ceramide, lactosylceramide, galactosylceramide, glucosylceramide, or any combination of two or more thereof. **Claim 7** The method according to any one of claims 1 to 5, wherein the sphingolipid is ceramide, lactosylceramide, galactosylceramide, or any combination of two or more thereof. **Claim 8** wherein the lipidoid is 306 Ｏｉ１０ ; 306O １０ ; 503O ｉ１０ ; 402O ６，１０ ; 500X １ ; 500O ｉ１０ ; 306O １１ ; 306Oi10; 306O １２ ; 200X ６ ; 516O ｉ１０ ; 500O １，１，８ ; 514X ６ ; 306O １４ ; 501X １ ; 205O １６ ; 500O １３ ; 113O ｉ１０ ; 306O １６ ; 306O １３ ; 205O １８ ; 509X ７ ; 501O ｉ１０ ; 503O ｉ１０ ; 500O １４ ; 113O ｉ１０ ; 509X １ ; 509X ３ ; 501X ２ ; 402O ６，１０ ; 516O ４，８ ; 402X ８ ; 501O １，１，８ ; or one or more of 509O １，１，８ The method according to any one of claims 1 to 7, which is one or more of the above. **Claim 9** wherein the lipidoid is 306 Ｏｉ１０ , 306O １０ , 503O ｉ１０ , and 402O ６，１０ The method according to any one of claims 1 to 7, which is one or more of the above. **Claim 10** wherein the ionizable lipidoid is 306 Ｏｉ１０ The method according to claim 1, wherein the ionizable lipidoid is 306 **Claim 11** The method according to any one of claims 1 to 10, wherein the therapeutic agent is anionic or polyanionic. **Claim 12** The method according to any one of claims 1 to 10, wherein the therapeutic agent is a nucleic acid. **Claim 13** The method according to claim 12, wherein the nucleic acid comprises RNA. **Claim 14** The method according to claim 13, wherein the RNA comprises mRNA. **Claim 15** The method according to claim 13, wherein the RNA comprises an RNAi reagent, dsRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA (gRNA), long non-coding RNA (lncRNA), base editing gRNA (beRNA), prime editing gRNA (pegRNA), or transfer RNA (tRNA). **Claim 16** The method according to claim 13, wherein the RNA comprises a gRNA, beRNA, or pegRNA, and an mRNA encoding Cas9 for base editing or prime editing, or a Cas9 fusion protein.

17. The method according to claim 12, wherein the nucleic acid encodes heme oxygenase 1 (HO1), brain-derived neurotrophic factor (BDNF), or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein.

18. The method according to claim 12, wherein the nucleic acid is DNA.

19. The lipid-containing particles are in mole percentages of the sphingolipid helper lipid, the cholesterol or its derivative, the PEG-based compound, and the ionizable lipidoid, 5 to 95 mol% of the sphingolipid helper lipid; 5 to 75 mol% of the cholesterol or its derivative; 0.1 to 50 mol% of the PEG-based compound; and 5 to 90 mol% of the ionizable lipidoid The method according to any one of claims 1 to 17, which is a lipid nanoparticle comprising.

20. The method according to any one of claims 1 to 19, wherein the cholesterol or its derivative is cholesterol.

21. The PEG-based compound is one or more of PEG-ceramide, PEG-DMG, PEG-PE, poloxamer, or DSPE carboxy PEG, and the PEG-based compound is C14PEG2000DMG, C15PEG2000DMG, C16PEG2000DMG, C18PEG2000DMG, C14PEG2000ceramide, C15PEG2000ceramide, C16PEG2000ceramide, C18PEG2000ceramide, C14PEG2000PE, C15PEG2000PE, C16PEG2000PE, C18PEG2000PE, C14PEG350PE, C14PEG5000PE, poloxamer F-127, poloxamer F-68, poloxamer L-64, and DSPE carboxy PEG. The method according to any one of claims 1 to 20.

22. The PEG-based compound is a PEGylated fatty acid, for example, PEGylated C １０ -C ２０ fatty acid-containing compound, for example, C １４ -PEG ２０００ -PE, and the method according to claim 21.

23. The method according to any one of claims 1 to 22, wherein the nucleic acid encodes heme oxygenase 1 (HO1) and / or brain-derived neurotrophic factor (BDNF) for the treatment of ischemia or ischemia / reperfusion injury, such as ischemic stroke, in the patient.

24. The method according to any one of claims 1 to 22, wherein the nucleic acid encodes a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein for the treatment of cancer, such as glioblastoma, in the patient.

25. A lipid-containing particle comprising a therapeutic agent, wherein: A sphingolipid helper lipid; Cholesterol or a derivative thereof; A PEG-based compound such as a PEG-containing polymer or a PEGylated fatty acid-containing compound; and An ionizable lipidoid The lipid-containing particle further comprising.

26. The lipid-containing particle according to claim 25, wherein the lipid-containing particle is a lipid nanoparticle.

27. The lipid-containing particle according to claim 25 or 26, wherein the sphingolipid is ceramide, sphingomyelin, ceramide phosphoethanolamine, sphingosylphosphorylcholine, sphingosine, sphingoglycolipid, or any combination of two or more thereof.

28. The lipid-containing particle according to claim 27, wherein the sphingolipid is ceramide, glucosylceramide, galactosylceramide, or lactosylceramide.

29. The lipid-containing particle according to any one of claims 25 to 28, wherein the sphingolipid is a CNS sphingolipid, a brain sphingolipid, ceramide, lactosylceramide, galactosylceramide, glucosylceramide, or any combination of two or more thereof.

30. The lipid-containing particle according to any one of claims 25 to 28, wherein the sphingolipid is ceramide, lactosylceramide, galactosylceramide, or any combination of two or more thereof.

31. wherein the lipidoid is 306 Ｏｉ１０ ; 306O １０ ; 503O ｉ１０ ; 402O ６，１０ ; 500X １ ; 500O ｉ１０ ; 306O １１ ; 306Oi10; 306O １２ ; 200X ６ ; 516O ｉ１０ ; 500O １，１，８ ; 514X ６ ; 306O １４ ; 501X １ ; 205O １６ ; 500O １３ ; 113O ｉ１０ ; 306O １６ ; 306O １３ ; 205O １８ ; 509X ７ ; 501O ｉ１０ ; 503O ｉ１０ ; 500O １４ ; 113O ｉ１０ ; 509X １ ; 509X ３ ; 501X ２ ; 402O ６，１０ ; 516O ４，８ ; 402X ８ ; 501O １，１，８ ; or 509O １，１，８ The lipid-containing particles according to any one of claims 25 to 30, which are one or more of the above.

32. wherein the lipidoid is 306 Ｏｉ１０ , 306O １０ , 503O ｉ１０ , and 402O ６，１０ ; the lipid-containing particle according to any one of claims 25 to 30, which is one or more of the foregoing

33. The ionizable lipidoid is 306O ｉ１０ The lipid-containing particle according to claim 25, wherein the ionizable lipidoid is 306O

34. The lipid-containing particle according to any one of claims 25 to 33, wherein the therapeutic agent is anionic or polyanionic.

35. The lipid-containing particle according to any one of claims 25 to 33, wherein the therapeutic agent is a nucleic acid.

36. The lipid-containing particle according to claim 35, wherein the nucleic acid comprises RNA.

37. The lipid-containing particle according to claim 36, wherein the RNA comprises mRNA.

38. The lipid-containing particle according to claim 36, wherein the RNA comprises an RNAi reagent, dsRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA (gRNA), long non-coding RNA (lncRNA), base editing gRNA (beRNA), prime editing gRNA (pegRNA), or transfer RNA (tRNA).

39. The lipid-containing particle according to claim 36, wherein the RNA comprises gRNA, beRNA, or pegRNA, and an mRNA encoding Cas9 for base editing or prime editing, or a Cas9 fusion protein.

40. The lipid-containing particle according to claim 35, wherein the nucleic acid encodes heme oxygenase 1 (HO1), brain-derived neurotrophic factor (BDNF), or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein.

41. The lipid-containing particle according to claim 35, wherein the nucleic acid is DNA.

42. The lipid-containing particle is in terms of mol% of the sphingolipid helper lipid, the cholesterol or its derivative, the PEG-based compound, and the ionizable lipidoid, 5 to 95 mol% of the sphingolipid helper lipid; 5 to 75 mol% of the cholesterol or its derivative; 0.1 to 50 mol% of the PEG-based compound; and 5 to 90 mol% of the ionizable lipidoid The lipid-containing particle according to any one of claims 25 to 41, which is a lipid nanoparticle containing the above components.

43. The lipid-containing particle according to any one of claims 25 to 42, wherein the cholesterol or its derivative is cholesterol.

44. The PEG-based compound is one or more of PEG-ceramide, PEG-DMG, PEG-PE, poloxamer, or DSPE carboxy PEG, and the PEG-based compound is C14PEG2000DMG, C15PEG2000DMG, C16PEG2000DMG, C18PEG2000DMG, C14PEG2000 ceramide, C15PEG2000 ceramide, C16PEG2000 ceramide, C18PEG2000 ceramide, C14PEG2000PE, C15PEG2000PE, C16PEG2000PE, C18PEG2000PE, C14PEG350PE, C14PEG5000PE, poloxamer F-127, poloxamer F-68, poloxamer L-64, and DSPE carboxy PEG. The lipid-containing particles according to any one of claims 25 to 43.

45. The PEG-based compound is a PEGylated fatty acid, for example, PEGylated C １０ -C ２０ fatty acid-containing compound, for example, C １４ -PEG ２０００ The lipid-containing particle according to claim 44, comprising -PE.

46. The nucleic acid encodes heme oxygenase 1 (HO1) and / or brain-derived neurotrophic factor (BDNF) for the treatment of ischemia or ischemia / reperfusion injury, such as ischemic stroke, in a patient. The lipid-containing particles according to any one of claims 25 to 45.

47. The nucleic acid encodes a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein for the treatment of cancer, such as glioblastoma, in a patient. The lipid-containing particles according to any one of claims 25 to 45.

48. The lipid-containing particles according to any one of claims 25 to 45 for the treatment of neuroinflammation, neurodegenerative diseases, or monogenic neuropathies.

49. A method of treating a patient suffering from ischemia, stroke, ischemia / reperfusion injury, neuroinflammation, neurodegenerative disease, monogenic neuropathy, or cancer, comprising administering to the patient an effective amount of the lipid nanoparticles according to any one of claims 25 to 45, thereby treating the ischemia, stroke, ischemia / reperfusion injury, neuroinflammation, neurodegenerative disease, monogenic neuropathy, or cancer in the patient.

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