WO2026022656A2 - Formulations de particules lipidiques contenant des lipides cationiques chargés en permanence - Google Patents

Formulations de particules lipidiques contenant des lipides cationiques chargés en permanence

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Publication number
WO2026022656A2
WO2026022656A2 PCT/IB2025/057322 IB2025057322W WO2026022656A2 WO 2026022656 A2 WO2026022656 A2 WO 2026022656A2 IB 2025057322 W IB2025057322 W IB 2025057322W WO 2026022656 A2 WO2026022656 A2 WO 2026022656A2
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WIPO (PCT)
Prior art keywords
lipid
mol
lipid nanoparticle
substituted
nanoparticle
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PCT/IB2025/057322
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English (en)
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WO2026022656A3 (fr
Inventor
James Heyes
Kieu Lam
Richard James Holland
Mark Christopher Wood
Stephen Reid
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Genevant Sciences GmbH
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Genevant Sciences GmbH
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Publication of WO2026022656A2 publication Critical patent/WO2026022656A2/fr
Publication of WO2026022656A3 publication Critical patent/WO2026022656A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • LNPs Lipid nanoparticles
  • siRNA small interfering RNA
  • mRNA messenger RNA
  • HSCs hepatic stellate cells
  • nucleic acids such as siRNA or mRNA can be delivered directly to these cells. These nucleic acids can modulate the gene expression within HSCs, potentially inhibiting the process of fibrogenesis and slowing or even reversing the progression of liver fibrosis and hepatocellular carcinoma. This targeted approach has the potential to provide a more precise treatment, potentially reducing off-target effects and improving the therapeutic index.
  • the disclosure provides a method for reducing expression of a gene in a hepatic stellate cell.
  • the method includes contacting the hepatic stellate cell with a lipid nanoparticle including a nucleic acid.
  • the nucleic acid is configured to reduce expression of the gene in the hepatic stellate cell.
  • the lipid nanoparticle further includes an ionizable lipid or a pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle further includes a phospholipid or a pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle further includes cholesterol or a derivative thereof.
  • the lipid nanoparticle further includes a conjugated lipid.
  • the lipid nanoparticle further includes a permanently charged cationic lipid or a pharmaceutically acceptable salt thereof.
  • the contacting of the hepatic stellate cell with the lipid nanoparticle introduces the nucleic acid into the hepatic stellate cell, thereby reducing expression of the gene in the hepatic stellate cell.
  • the disclosure provides a lipid nanoparticle.
  • the lipid nanoparticle includes a nucleic acid.
  • the lipid nanoparticle further includes an ionizable lipid or a pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle further includes a phospholipid or a pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle further includes cholesterol or a derivative thereof.
  • the lipid nanoparticle further includes a conjugated lipid.
  • the lipid nanoparticle further includes a permanently charged cationic lipid or a pharmaceutically acceptable salt thereof.
  • the permanently charged cationic lipid includes one or more quaternary ammonium nitrogens.
  • the permanently charged cationic lipid further includes a heteroatom branch point other than the one or more quaternary ammonium nitrogens.
  • the heteroatom branch point is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • the permanently charged cationic lipid further includes three or more terminal hydrocarbon chains not directly bonded to the one or more quaternary ammonium nitrogens. The terminal hydrocarbon chains are each independently alkyl or alkenyl.
  • the disclosure provides another lipid nanoparticle.
  • the lipid nanoparticle includes a nucleic acid.
  • the lipid nanoparticle further includes an ionizable lipid or a pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle further includes a phospholipid or a pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle further includes cholesterol or a derivative thereof.
  • the lipid nanoparticle further includes a conjugated lipid.
  • the lipid nanoparticle further includes a permanently charged cationic lipid or a pharmaceutically acceptable salt thereof.
  • the permanently charged cationic lipid includes one or more quaternary ammonium nitrogens.
  • the permanently charged cationic lipid further includes two or more carbon branch points, each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkoxy, alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • the permanently charged cationic lipid further includes three or more terminal hydrocarbon chains not directly bonded to the one or more quaternary ammonium nitrogens.
  • the terminal hydrocarbon chains are each independently alkyl or alkenyl.
  • the disclosure provides another lipid nanoparticle.
  • the lipid nanoparticle includes a nucleic acid.
  • the lipid nanoparticle further includes an ionizable lipid or a pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle further includes a phospholipid or a pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle further includes a nucleic acid.
  • the lipid nanoparticle further includes an i
  • 3 78645288V.1 includes cholesterol or a derivative thereof.
  • the lipid nanoparticle further includes a conjugated lipid.
  • the lipid nanoparticle further includes a permanently charged cationic lipid or a pharmaceutically acceptable salt thereof.
  • the permanently charged cationic lipid includes one or more quaternary ammonium nitrogens.
  • the permanently charged cationic lipid further includes one or more hydrolyzable linkages. Each of the one or more hydrolyzable linkages is independently a silyl ether linkage, a siloxane linkage, a disulfide linkage, a carbonate linkage, a pyrrolidine dicarboxylate linkage, or a cyclic benzylidene acetal linkage.
  • the disclosure provides a composition, e.g., a pharmaceutical composition.
  • the pharmaceutical composition includes a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient.
  • the pharmaceutical composition further includes a lipid nanoparticle as disclosed herein or a compound as disclosed herein.
  • the disclosure provides an in vivo method of delivering a nucleic acid to a subject. The method includes administering to the subject a lipid nanoparticle as disclosed herein, or a pharmaceutical composition including a lipid nanoparticle as disclosed herein.
  • the disclosure provides a method of preventing or treating a disease or disorder in a subject.
  • FIG. 1 presents a graph plotting Reelin (RELN) mRNA expression normalized with respect to mRNA expression for the housekeeping gene GAPDH in mice administered various dosage concentrations of lipid nanoparticles including RELN siRNA and having a composition according to a provided embodiment.
  • the graph further includes expression data for mice administered a phosphate buffered saline (PBS) control.
  • PBS phosphate buffered saline
  • FIG. 2 presents a graph plotting the alanine transaminase (ALT) and aspartate aminotransferase (AST) activities in the mice of FIG.1 administered 3 mg/kg or 6 mg/kg lipid nanoparticle doses, or administered PBS control.
  • FIG 3 presents a graph plotting body weight (BW) change over time for the mice of FIG.1 administered 6 mg/kg lipid nanoparticle doses, or administered PBS control.
  • BW body weight
  • FIG. 4 presents a graph plotting Reelin (RELN) mRNA expression normalized with respect to mRNA expression for the housekeeping gene GAPDH in whole liver lysates of mice administered various dosage concentrations of lipid nanoparticles including RELN siRNA and having a composition including a quaternary salt lipid according to a provided embodiment.
  • RELN Reelin
  • TTR Transthyretin
  • lipid nanoparticles are beneficially effective in targeting hepatic stellate cells with enhanced efficiency.
  • the efficiency of lipid nanoparticles in delivering nucleic acids to certain cell types can be very important for many therapeutic applications, particularly when the nucleic acids delivered by these nanoparticles are used for silencing or reducing the expression of a gene in the targeted cell type.
  • High-efficiency delivery can enhance the likelihood that a significant portion of the nucleic acids carried by the lipid nanoparticles reach their intended destination, thereby increasing the therapeutic potential of those nucleic acids.
  • inefficient delivery can lead to insufficient alteration of the gene expression to be silenced or reduced.
  • the benefits of enhanced delivery efficiency can include not only improved effectiveness of the treatment, but also reductions to the amount of lipid nanoparticles and/or nucleic acids needed to produce the desired treatment effect, thereby minimizing potential toxicity and side effects.
  • the materials and methods disclosed herein address these challenges by using particular lipid nanoparticle formulations that generally include a permanently charged
  • the provided lipid nanoparticle formulations include selected species of these five lipid components combined in particular relative amounts to yield lipid nanoparticles that are surprisingly efficient in targeting desired cell types, such as hepatic stellate cells. Furthermore, the lipid nanoparticles can be advantageously useful in therapies involving the down-regulation (e.g., the silencing or reducing) of gene expression in these targeted cells (e.g., hepatic stellate cells).
  • the compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds.
  • the compounds may be labeled with isotopes, such as for example deuterium ( 2 H), tritium ( 3 H), iodine-125 ( 125 I), carbon-13 ( 13 C), or carbon-14 ( 14 C).
  • substitution when used in relation to a chemical substance, refers to replacement of a hydrogen atom with a non-hydrogen atom or covalently bonded group of atoms.
  • the atom or group of atoms replacing the hydrogen atom is referred to as a “substituent.”
  • member when used in relation to a chemical substance, refers to a non-hydrogen atom of a covalently bonded group of atoms, e.g., a compound or substituent thereof.
  • references herein to a phospholipid optionally includes a combination of two or more phospholipids, and the like.
  • references herein to a plurality (i.e., two or more) of a material or a population of a material can indicate that multiple units of the material are present, where the multiple units are copies (e.g., identical or essentially identical copies) of one another.
  • references to a plurality (i.e., two or more) of a material or a population of a material can indicate that multiple species of the material are present, where the multiple species can themselves each independently be represented by a single unit or by multiple units.
  • reference to two or more phospholipids can indicate that multiple molecules of the same phospholipid compound are present, or can indicate that multiple different types of phospholipid compounds are present, each independently in the form of one or more individual molecules.
  • the term “between,” when used in the context of a range of values, indicates that the range includes the upper and lower values of the range. Thus, for example, reference to a value that is “between” 1 and 10 indicates that the value may be 1, the value may be 10, or the value may be greater than 1 and less than 10. Stated differently, a value that is “between” 1 and 10 is a value that is greater than or equal to 1 and is less than or equal to 10. [0031] As used herein, the term “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
  • the terms “including,” “comprising,” “having,” “containing,” and variations thereof, are inclusive and open-ended and do not exclude additional, unrecited elements or method steps beyond those explicitly recited.
  • the phrase “consisting of” is closed and excludes any element, step, or ingredient not explicitly specified.
  • the phrase “consisting essentially of” limits the scope of the described feature to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the disclosed feature.
  • the term “optional” or “optionally” means that the subsequently described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
  • lipid particle refers to a particle comprising a phospholipid and an ionizable lipid.
  • a lipid particle may comprise additional lipid components, such as a sterol and/or a conjugated lipid, and may further comprise a nucleic acid, where the nucleic acid may be encapsulated within the particle.
  • phospholipid refers to a lipid species having a phosphate- containing hydrophilic “head group” and a hydrophobic moiety.
  • the hydrophobic moiety can be any hydrophobic moiety.
  • 8 78645288V.1 comprise one or more hydrophobic groups, most typically two hydrophobic groups.
  • the hydrophobic groups are also referred to as hydrophobic “tails,” and can be derived from fatty acids and joined by an alcohol residue, e.g., glycerol.
  • Exemplary structures of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine.
  • ionizable lipid refers to a lipid species characterized by the presence of one or more functional groups that can acquire a net positive charge depending on the surrounding environmental pH. These lipids typically contain ionizable amine, carboxylate, or phosphate groups, or other pH-sensitive moieties that enable reversible protonation or deprotonation. Ionizable lipids may be engineered to remain neutral or minimally charged at physiological pH (approximately 7.4), promoting reduced systemic toxicity and improved biocompatibility, while becoming protonated and/or to have a net positive charge in acidic environments, such as those encountered in endosomal or lysosomal compartments.
  • an ionizable lipid includes an ionizable primary, secondary, or tertiary amine (e.g., pH titratable) head group.
  • ionizable lipids promote encapsulation of a negatively charged nucleic acid (e.g., mRNA or siRNA) payload during particle formation.
  • ionizable lipids promote endosomal fusion and cytoplasmic release of a payload following cellular uptake of a lipid nanoparticle.
  • the terms “permanently charged lipid,” “permanently charged cationic lipid,” and “permanently charged ionic lipid” generally refer to a lipid species that carries a net positive charge regardless of the pH of its biological environment.
  • conjugated lipid refers to a polymer-conjugated lipid, e.g., a polymer-conjugated lipid that inhibits aggregation of lipid particles.
  • Such lipid conjugates include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), polysarcosine-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, PEG-lipid conjugates, such as PEG coupled to dialkyloxypropyls, PEG coupled to diacylglycerols, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, PEG conjugated to ceramides (see, e.g., U.S. Pat. No.5,885,613, the disclosure of which is herein incorporated by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof.
  • PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable
  • 9 78645288V.1 for coupling the polymer to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties.
  • number average molecular weight and the abbreviation “Mn,” when used to describe a characteristic of a polymer, refer to a molecular weight measurement that is calculated by dividing the total weight of all the polymer molecules in a sample with the total number of polymer molecules in the sample.
  • salt refers to acid or base salts of the compounds of the present disclosure.
  • a “pharmaceutically acceptable salt” is one that is compatible with other ingredients of a formulation composition containing the compound, and that is not deleterious to a recipient thereof, i.e., a subject. It is thus understood that the pharmaceutically acceptable salts do not cause a significant adverse toxicological effect on the subject.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form.
  • nucleic acids containing nucleotide analogs or modified backbone residues or linkages which are synthetic, naturally occurring, and non- naturally occurring, that have similar binding properties as the reference nucleotide and are metabolized in a manner similar to reference nucleotides.
  • nucleotide analogs are described in, e.g., International Patent Application No. WO 2007/024708.
  • nucleic acids having such nucleotide analogs, modified backbone residues, or linkages include, without limitation, those containing phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2′-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).
  • PNAs peptide nucleic acids
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine (Batzer et al., (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605; and Rossolini et al., (1994) Mol. Cell. Probes 8:91).
  • Non-limiting examples of polynucleotides or nucleic acids include DNA, RNA, coding or noncoding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA),
  • ribosomal RNA rRNA
  • siRNA short interfering RNA
  • shRNA short-hairpin RNA
  • miRNA micro- RNA
  • small nucleolar RNA(snoRNA) ribozymes
  • dNTPs deoxynucleotides
  • ddNTPs dideoxynucleotides
  • Polynucleotides can also include complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification.
  • cDNA complementary DNA
  • Polynucleotides can also include DNA molecules produced synthetically or by amplification, genomic DNA (gDNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, or primers.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides, nucleotide analogs, and/or nucleosides suitable for reducing the immunogenicity of RNA, such as those described in International Patent Application No. WO 2007/024708. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer.
  • nucleic acids or polynucleotides can be double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive, for example, a double-stranded nucleic acid need not be double-stranded along the entire length of both strands.
  • Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole.
  • Such modifications include base modifications such as 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitutions of S-bromo-uracil, backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like.
  • Nucleic acid(s) can be derived from a completely chemical synthesis process, such as a solid phase-mediated chemical synthesis, from a biological source, such as through isolation from any species that produces nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, in vitro transcription such as described in, e.g., International Patent Application Publication No. WO 2007/024708, or from a combination of those processes.
  • a completely chemical synthesis process such as a solid phase-mediated chemical synthesis
  • a biological source such as through isolation from any species that produces nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, in vitro transcription such as described in, e.g., International Patent Application Publication No. WO 2007/024708, or from a combination of those processes.
  • the term “operably linked” refers to a functional linkage between a first nucleic acid sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the first nucleic acid sequence affects the transcription, translation, or stability of the second nucleic acid sequence
  • the term “alkyl,” by itself or as part of another substituent refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated.
  • a branched alkyl may include one or more branches having a geminal, vicinal, and/or isolated pattern.
  • an alkyl may include gem-methyl groups.
  • Alkyl may include any number of carbons, such as C 1-2 , C 1-3 , C 1-4 , C 1-5 , C 1-6 , C 1-7 , C 1-8 , C 1-9 , C 1-10 , C 2-3 , C 2-4 , C 2-5 , C 2-6 , C 3-4 , C3-5, C3-6, C4-5, C4-6 and C5-6.
  • C1-6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc.
  • Alkyl may also refer to alkyl groups having up to 40 carbons atoms or more, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. In some aspects, alkyl groups may be substituted. Unless otherwise specified, “substituted alkyl” groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy. [0049] As used herein, the term “alkenyl,” by itself or as part of another substituent, refers to a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one carbon- carbon double bond.
  • Alkenyl may include any number of carbons, such as C2, C2-3, C2-4, C2-5, C2-6, C2-7, C2-8, C2-9, C2-10, C3, C3-4, C3-5, C3-6, C4, C4-5, C4-6, C5, C5-6, and C6.
  • Alkenyl groups may have any suitable number of double bonds, including, but not limited to, 1, 2, 3, 4, 5 or more.
  • alkenyl groups include, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, and 1,3,5-hexatrienyl.
  • alkenyl groups may be substituted.
  • substituted alkenyl groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy.
  • alkoxy refers to a substituted alkyl group, as defined above, having an oxygen atom that connects the alkyl group to the point of attachment: alkyl-O-.
  • alkoxy groups may have any suitable number of carbon atoms, such as C 1-6 .
  • Alkoxy groups include,
  • alkenoxy refers to a substituted alkenyl group, as defined above, having an oxygen atom that connects the alkenyl group to the point of attachment: alkenyl-O-.
  • alkenoxy groups may have any suitable number of carbon atoms, such as C1-6.
  • heteroalkyl refers to an alkyl group of any suitable length and having any number of heteroatoms selected from N, O and S.
  • the heteroatoms may also be oxidized, such as, but not limited to, -S(O)- and -S(O) 2 -.
  • heteroalkyl may include ethers, thioethers and alkyl-amines.
  • the heteroatom portion of the heteroalkyl may replace a hydrogen of the alkyl group to form a hydroxy, thio, or amino group.
  • the heteroatom portion may be the connecting atom, or be inserted between two carbon atoms.
  • heteroalkyl groups may be substituted. Unless otherwise specified, “substituted heteroalkyl” groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy.
  • heteroalkenyl by itself or as part of another substituent, refers to an alkenyl group of any suitable length and having any number of heteroatoms selected from N, O, and S. The heteroatoms may also be oxidized, such as, but not limited to, -S(O)- and -S(O)2-.
  • heteroalkenyl may include ethers, thioethers and alkyl-amines.
  • the heteroatom portion of the heteroalkenyl may replace a hydrogen of the alkyl group to form a hydroxy, thio, or amino group.
  • the heteroatom portion may be the connecting atom, or be inserted between two carbon atoms.
  • heteroalkenyl groups may be substituted.
  • substituted heteroalkenyl groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, oxo, nitro, cyano, and alkoxy.
  • halo and halogen, by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.
  • heteroatom refers to a non-carbon atom replacing a carbon atom of a covalently bonded group of atoms, e.g., any of the functional groups described herein.
  • branch point when used in the context of a chemical compound, structure, or formula, refers to an atom that is not a member of a ring and that is directly covalently bonded to three or more non-hydrogen atoms.
  • hydrolyzable linkage refers to a functional group that can be cleaved by water, optionally through an enzymatically catalyzed reaction, under physiological conditions.
  • the term “fully encapsulated” indicates that the nucleic acid in the particles is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free nucleic acids.
  • a fully encapsulated system preferably less than 25% of particle nucleic acid is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than 10% and most preferably less than 5% of the particle nucleic acid is degraded.
  • Fully encapsulated also suggests that the particles are serum stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration.
  • the term “contacting” refers to either the direct or indirect in vitro or in vivo delivering of lipid nanoparticles to the surfaces of cells, e.g., by providing lipid nanoparticles at or proximate to a location of the cells to be contacted.
  • the in vitro contacting may involve, for example, cells in a cell culture or tissue culture.
  • the cell culture or tissue culture may include cells in a suspension and/or adherent cells.
  • the contacted cells may be of same cell type or of two or more different cell types. Merely for illustration, different contacted cell types may include, for example, hepatocytes and hepatic stellate cells.
  • different cell types are cultured together before and/or during the contacting with lipid nanoparticles.
  • different cell types are cultured separately.
  • one cell type of two or more cell types to be contacted is specifically cultured in the absence of any other cell types of the two or more cell types.
  • hepatic stellate cells are cultured, either alone or in combination with other cell types from the liver, such as, e.g., liver cells, prior to and/or during the contacting.
  • the tissue culture is a liver tissue culture.
  • the in vivo contacting of cells typically involves administering the lipid nanoparticles to a subject, where the cells are within the body of the subject. In some examples, the administering is proximate
  • the term “subject” refers to a vertebrate, and preferably to a mammal. Mammalian subjects for which the provided composition is suitable include, but are not limited to, mice, rats, simians, humans, farm animals, sport animals, and pets. In some embodiments, the subject is human. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject is an adult.
  • the subject is an adolescent. In some embodiments, the subject is a child. In some embodiments, the subject is above 10 years of age, e.g., above 20 years of age, above 30 years of age, above 40 years of age, above 50 years of age, above 60 years of age, above 70 years of age, or above 80 years of age. In some embodiments, the subject is less than 80 years of age, e.g., less than 70 years of age, less than 60 years of age, less than 50 years of age, less than 40 years of age, less than 30 years of age, less than 20 years of age, or less than 10 years of age.
  • the terms “pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and may be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the subject.
  • pharmaceutically acceptable excipients and carriers include water, NaCl, normal saline solutions, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, and the like.
  • pharmaceutically acceptable excipients and carriers include water, NaCl, normal saline solutions, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, and the like.
  • administering refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous, intrathecal, intracerebroventricular, intraparenchymal, subretinal, or intravitreal administration, or the implantation of a slow-release device e.g., a mini-osmotic pump, to the subject.
  • a slow-release device e.g., a mini-osmotic pump
  • the terms “treat”, “treating” and “treatment” refers to a procedure resulting in any indicia of success in the elimination or amelioration of an injury, pathology, condition, or symptom (e.g., pain), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of one or more symptoms.
  • the present disclosure provides various lipid nanoparticles that generally include five distinct lipid components: an ionizable lipid or a pharmaceutically acceptable salt thereof, a phospholipid or a pharmaceutically acceptable salt thereof, cholesterol or a derivative thereof, a conjugated lipid, and a permanently charged cationic lipid or a pharmaceutically acceptable salt thereof.
  • the provided lipid nanoparticles generally further include a nucleic acid.
  • compositions, and amounts (e.g., relative amounts) of these components provide surprising improvements in various properties of the lipid nanoparticles, e.g., properties advantageous for the delivery of the nucleic acid.
  • improved properties include, for example, enhanced targeting of hepatic stellate cells, decreased toxicity, good biodegradability, and high biocompatibility.
  • the lipid nanoparticles are demonstrated herein as being beneficially effective in delivering a nucleic acid to hepatic stellate cells when the permanently charged cationic lipid includes a quaternary ammonium nitrogen, and further includes (1) a heteroatom branch point other than the one or more quaternary ammonium nitrogens, the heteroatom branch point directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members; and three or more terminal hydrocarbon chains not directly bonded to the one or more quaternary ammonium nitrogens, where the terminal hydrocarbon chains are each independently alkyl or alkenyl; (2) two or more carbon branch points, each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkoxy,
  • the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between about 39 mol% and about 49 mol% of the total lipid of the lipid nanoparticle (e.g., between about 39 mol% and about 46.5 mol%, between about 39 mol% and about 44 mol%, between about 39 mol% and about 41.5 mol%, between about 41.5 mol% and about 49 mol%, between about 41.5 mol% and about 44 mol%, between about 44 mol% and about 49 mol%, between about 44 mol% and about 46.5 mol%, or between about 46.5 mol% and about 49 mol%)
  • the phospholipid or the pharmaceutically acceptable salt thereof comprises between about 8 mol% and about 12 mol% of the total lipid of the lipid nanoparticle (e.g., between about 8 mol% and about 11 mol%, between about 8 mol% and about 10 mol%, between about 8 mol% and about 9 mol%,
  • 17 78645288V.1 between about 1 mol% and about 11 mol% of the total lipid of the lipid nanoparticle (e.g., between about 1 mol% and about 8.5 mol%, between about 1 mol% and about 6 mol%, between about 1 mol% and about 3.5 mol%, between about 3.5 mol% and about 11 mol%, between about 3.5 mol% and about 8.5 mol%, between about 3.5 mol% and about 6 mol%, between about 6 mol% and about 11 mol%, between about 6 mol% and about 8.5 mol%, or between about 8.5 mol% and about 11 mol%).
  • the total lipid of the lipid nanoparticle e.g., between about 1 mol% and about 8.5 mol%, between about 1 mol% and about 6 mol%, between about 1 mol% and about 3.5 mol%, between about 3.5 mol% and about 11 mol%, between about 3.5 mol% and about 8.5 mol%,
  • the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 39 mol% and 49 mol% of the total lipid of the lipid nanoparticle (e.g., between about 39 mol% and about 46.5 mol%, between about 39 mol% and about 44 mol%, between about 39 mol% and about 41.5 mol%, between about 41.5 mol% and about 49 mol%, between about 41.5 mol% and about 44 mol%, between about 44 mol% and about 49 mol%, between about 44 mol% and about 46.5 mol%, or between about 46.5 mol% and about 49 mol%)
  • the phospholipid or the pharmaceutically acceptable salt thereof comprises between about 9 mol% and about 11 mol% of the total lipid of the lipid nanoparticle (e.g., between about 9 mol% and about 10.5 mol%, between about 9 mol% and about 10 mol%, between about 9 mol% and about 9.5 mol%,
  • the present disclosure also provides a population of lipid nanoparticles, where the population includes any of the lipid nanoparticles disclosed herein.
  • the population includes or consists of multiple lipid nanoparticles that each have an identical or similar formulation, e.g., that were each prepared from and/or that have an identical or essentially identical composition of lipids and, optionally, other components.
  • the identity and relative amounts of the lipid nanoparticle components can also be selected to provide the lipid nanoparticle with a size that is beneficial for use in delivering a nucleic acid, e.g., to a hepatic stellate cell.
  • the mean diameter of a population of the lipid nanoparticles described herein is reported as a Z-average value.
  • Z-average in the context of a mean diameter of a population of lipid nanoparticles, refers to the intensity-weighted mean hydrodynamic diameter calculated using dynamic light scattering (DLS) techniques.
  • DLS dynamic light scattering
  • the Z-average is derived from a cumulants analysis of the autocorrelation function of scattered light intensity, and is calculated according to approaches described in, for example, International Organization for Standardization (ISO) Standard No.22412 (2017), and Thomas, J. Colloid Interface Sci.117, (1987): 187.
  • the mean diameter of a population of the lipid nanoparticles described herein is calculated using dynamic light scattering measurements determined at a backscatter angle of 173 degrees with a Malvern Zetasizer instrument (Malvern Panalytical Ltd, Malvern, UK).
  • a Malvern Zetasizer instrument Malvern Panalytical Ltd, Malvern, UK.
  • the lipid nanoparticles have a composition and/or are produced using a method according to descriptions in this disclosure.
  • the lipid nanoparticles can have a mean diameter that is, for example, between about 40 nm and about 150 nm, between about 50 nm and about 150 nm, between about 60 nm and about 130 nm, between about 70 nm and about 110 nm, between about 60 nm and about 100 nm, between about 50 and about 80 nm, between about 60 and about 80 nm, between about 60 and about 90 nm, or between about 70 and about 90 nm.
  • the specific configuration of the lipid nanoparticles described herein can also provide the nanoparticles with a pKa giving the nanoparticles targeting, stability, degradability, and toxicity properties advantageous for, e.g., the delivery of nucleic acids.
  • the lipid nanoparticle has a pKa between about 6.4 and about 6.9, e.g., between about 6.4 and about 6.8, between about 6.4 and about 6.7, between about 6.4 and about 6.6, between about 6.4 and about 6.5, between about 6.5 and about 6.9, between about 6.5 and about 6.8, between about 6.5 and about 6.7, between about 6.5 and about 6.6, between about 6.6 and about 6.9, between about 6.6 and about 6.8, between about 6.6 and about 6.7, between about 6.7 and about 6.9, between about 6.7 and about 6.8, or between about 6.8 and about 6.9.
  • a pKa between about 6.4 and about 6.9, e.g., between about 6.4 and about 6.8, between about 6.4 and about 6.7, between about 6.4 and about 6.6, between about 6.4 and about 6.5, between about 6.5 and about 6.9, between about 6.5 and about 6.9, between about 6.7 and about 6.8, or between about 6.8 and about 6.9.
  • the lipid nanoparticle pKa can be, for example, no more than about 6.7, e.g., no more than about 6.8, no more than about 6.7, no more than about 6.6, or no more than about 6.5. In terms of lower limits, the lipid nanoparticle pKa can be, for example, no less than about 6.4, e.g., no less than about 6.5, no less than about 6.6, no less than about 6.7, or no less than about 6.8. [0072] In a preferred embodiment, the pKa of the lipid nanoparticle described herein is determined using an assay measuring ionization at a temperature between about 34 °C and about 40 °C.
  • the lipid nanoparticle has a pKa, as determined using an assay performed at a temperature between about 34 °C and about 40 °C, that is from about 5.6 to about 7.4, or within any of the pKa ranges listed above.
  • the pKa of the lipid nanoparticle described herein is determined using an assay measuring ionization at a temperature of about 37 °C.
  • the lipid nanoparticle has a pKa, as determined using an assay performed at a temperature of about 37 °C, that is from about 6.4 to about 6.9, or within any of the pKa ranges listed above.
  • the pKa of the lipid nanoparticle described herein is determined using an assay measuring ionization at more than 5 different pH levels.
  • the lipid nanoparticle has a pKa, as determined using an assay performed at more than 5 different pH levels, that is from about 5.6 to about 7.4, or within any of the pKa ranges listed above.
  • the pKa of the lipid nanoparticle described herein is determined using an assay measuring ionization at more than 15 different pH levels.
  • the lipid nanoparticle has a pKa, as determined using an assay performed at more than 15 different pH levels, that is from about 5.6 to about 7.4, or within any of the pKa ranges listed above.
  • the pKa of the provided lipid nanoparticle is determined using in situ fluorescence titration, and preferably TNS fluorescence titration.
  • This assay typically yields a sigmoidal curve showing fluorescence, such as TNS fluorescence, wherein the pKa of the provided lipid nanoparticle is determined to be the value that corresponds to 0.5 normalized fluorescence, such as normalized TNS fluorescence, on a scale from 0 to 1.0.
  • the pKa of the provided lipid nanoparticle is determined using the assay described in Example 8.
  • the lipid nanoparticle has a pKa, as determined using the assay described in Example 8, that is from about 6.4 to about 6.9, or within any of the pKa ranges listed above.
  • the provided lipid nanoparticles can have in vivo clearance and degradation characteristics that are particularly beneficial when the nanoparticle is used to administer a nucleic acid to a subject, e.g., to provide a treatment to the subject and/or to silence genetic information of the subject. Clearance and degradation rates that are too high can prevent a lipid nanoparticle from protecting a cargo, such as a nucleic acid cargo, and from successfully delivering it to its targeted destination within the subject.
  • the provided lipid nanoparticles also can exhibit advantageous specificity in delivering, e.g., nucleic acids, to a targeted site, e.g., hepatic stellate cells, in a subject when the lipid nanoparticle is administered to the subject.
  • the provided lipid nanoparticles can further be configured, e.g., through the selection of their components, to be substantially non- toxic.
  • A. Permanently Charged Cationic lipid [0076]
  • the provided lipid nanoparticle generally includes at least one permanently charged cationic lipid or a pharmaceutically acceptable salt thereof, where the permanently charged cationic lipid includes one or more quaternary ammonium nitrogens.
  • the permanently charged cationic lipid can be selected or designed to provide the lipid nanoparticle with a desired particle size, stability, and capacity for encapsulation of nucleic acids. Further, lipid nanoparticles including the particular permanently charged cationic lipids described herein exhibit, for example, enhanced targeting, optimized clearance rates, and decreased toxicity and immune stimulation. In some embodiments, the lipid nanoparticle includes one species of permanently charged cationic lipid. In other embodiments, the lipid nanoparticle includes two or more species of permanently charged cationic lipids, e.g., three or more, four or more, five
  • the permanently charged cationic lipid of the provided lipid nanoparticle includes a heteroatom branch point other than the one or more quaternary ammonium nitrogens.
  • the permanently charged cationic lipid includes two heteroatom branch points other than the quaternary ammonium nitrogens.
  • the permanently charged cationic lipid includes three or more heteroatom branch points other than the quaternary ammonium nitrogens.
  • the heteroatoms of the one or more heteroatom branch points other than the quaternary ammonium nitrogens can be identical to one another, or can be different.
  • the branch point heteroatoms other than the quaternary ammonium nitrogens can include or consist of, for example, nitrogen and/or silicon.
  • at least one of the heteroatom branch points other than the quaternary ammonium nitrogens is directly bonded to three or more groups independently selected from alkyl, alkoxy, alkenoxy, heteroalkyl, and heteroalkenyl groups, each independently having five or more members, and each optionally oxo-substituted and optionally halogen-substituted.
  • At least one of the heteroatom branch points (other than a quaternary ammonium nitrogen) of the permanently charged cationic lipid of the provided lipid nanoparticle is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, alkenoxy, or heteroalkyl groups, each independently having five or more members.
  • at least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, alkenoxy, or heteroalkenyl groups, each independently having five or more members.
  • At least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members. In some embodiments, at least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenoxy, heteroalkyl or heteroalkenyl groups, each independently having five or more members.
  • At least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkoxy, alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • At least one of the heteroatom branch points (other than a quaternary ammonium nitrogen) of the permanently charged cationic lipid of the provided lipid nanoparticle is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, or alkenoxy groups, each independently having five or more members.
  • at least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, or heteroalkyl groups, each independently having five or more members.
  • At least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, or heteroalkenyl groups, each independently having five or more members. In some embodiments, at least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenoxy, or heteroalkyl groups, each independently having five or more members.
  • At least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen- substituted alkyl, alkenoxy, or heteroalkenyl groups, each independently having five or more members. In some embodiments, at least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • At least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkoxy, alkenoxy, or heteroalkyl groups, each independently having five or more members. In some embodiments, at least one of the heteroatom branch points is directly bonded to three or more optionally oxo- substituted and optionally halogen-substituted alkoxy, alkenoxy, or heteroalkenyl groups, each independently having five or more members.
  • At least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members. In some embodiments, at least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • At least one of the heteroatom branch points (other than a quaternary ammonium nitrogen) of the permanently charged cationic lipid of the provided lipid nanoparticle is directly bonded to three or more optionally oxo-substituted and optionally
  • At least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl or alkenoxy groups, each independently having five or more members.
  • at least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl or heteroalkyl groups, each independently having five or more members.
  • At least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl or heteroalkenyl groups, each independently having five or more members. In some embodiments, at least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkoxy or alkenoxy groups, each independently having five or more members. In some embodiments, at least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkoxy or heteroalkyl groups, each independently having five or more members.
  • At least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkoxy or heteroalkenyl groups, each independently having five or more members. In some embodiments, at least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenoxy or heteroalkyl groups, each independently having five or more members.
  • At least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenoxy or heteroalkenyl groups, each independently having five or more members. In some embodiments, at least one of the heteroatom branch points is directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted heteroalkyl or heteroalkenyl groups, each independently having five or more members.
  • At least one of the three or more groups directly bonded to a heteroatom branch point (other than a quaternary ammonium nitrogen) of the permanently charged cationic lipid of the provided lipid nanoparticle is an optionally oxo-substituted and optionally halogen substituted alkyl group having five or more members.
  • at least two of the three or more groups directly bonded to a heteroatom branch of the permanently charged cationic lipid of the lipid nanoparticle are each independently an optionally oxo-substituted and optionally halogen substituted alkyl group having five or more members.
  • a heteroatom branch of the permanently charged cationic lipid of the lipid nanoparticle are each independently an optionally oxo-substituted and optionally halogen substituted alkyl group having five or more members.
  • at least one of the three or more groups directly bonded to a heteroatom branch of the permanently charged cationic lipid of the provided lipid nanoparticle is an optionally oxo-substituted and optionally halogen substituted alkoxy group having five or more members.
  • At least two of the three or more groups directly bonded to a heteroatom branch of the permanently charged cationic lipid of the lipid nanoparticle are each independently an optionally oxo-substituted and optionally halogen substituted alkoxy group having five or more members.
  • at least three of the three or more groups directly bonded to a heteroatom branch of the permanently charged cationic lipid of the lipid nanoparticle are each independently an optionally oxo-substituted and optionally halogen substituted alkoxy group having five or more members.
  • At least one of the three or more groups directly bonded to a heteroatom branch of the permanently charged cationic lipid of the provided lipid nanoparticle is an optionally oxo-substituted and optionally halogen substituted alkenoxy group having five or more members.
  • at least two of the three or more groups directly bonded to a heteroatom branch of the permanently charged cationic lipid of the lipid nanoparticle are each independently an optionally oxo-substituted and optionally halogen substituted alkenoxy group having five or more members.
  • At least three of the three or more groups directly bonded to a heteroatom branch of the permanently charged cationic lipid of the lipid nanoparticle are each independently an optionally oxo-substituted and optionally halogen substituted alkenoxy group having five or more members.
  • at least one of the three or more groups directly bonded to a heteroatom branch of the permanently charged cationic lipid of the provided lipid nanoparticle is an optionally oxo-substituted and optionally halogen substituted heteroalkyl group having five or more members.
  • At least two of the three or more groups directly bonded to a heteroatom branch of the permanently charged cationic lipid of the lipid nanoparticle are each independently an optionally oxo-substituted and optionally halogen substituted heteroalkyl group having five or more members. In some embodiments, at least three of the three or more groups directly bonded to a heteroatom branch of the permanently charged cationic lipid of the lipid nanoparticle are each independently an optionally oxo-substituted and optionally halogen substituted heteroalkyl group having five or more members. In some embodiments, at least three of the three or more groups directly bonded to a heteroatom branch of the permanently
  • 25 78645288V.1 charged cationic lipid of the lipid nanoparticle are each independently an optionally oxo- substituted and optionally halogen substituted heteroalkyl group having five or more members.
  • at least one of the three or more groups directly bonded to a heteroatom branch of the permanently charged cationic lipid of the provided lipid nanoparticle is an optionally oxo-substituted and optionally halogen substituted heteroalkenyl group having five or more members.
  • At least two of the three or more groups directly bonded to a heteroatom branch of the permanently charged cationic lipid of the lipid nanoparticle are each independently an optionally oxo-substituted and optionally halogen substituted heteroalkenyl group having five or more members.
  • at least three of the three or more groups directly bonded to a heteroatom branch of the permanently charged cationic lipid of the lipid nanoparticle are each independently an optionally oxo- substituted and optionally halogen substituted heteroalkenyl group having five or more members.
  • the permanently charged cationic lipid of the provided lipid nanoparticle can include two or more carbon branch points, e.g., three or more carbon branch points, four or more carbon branch points, or five or more carbon branch points, that are each independently directly bonded to three or more groups independently selected from alkyl, alkoxy, alkenoxy, heteroalkyl, and heteroalkenyl groups, each independently having five or more members, and each optionally oxo-substituted and optionally halogen-substituted.
  • two or more carbon branch points e.g., three or more carbon branch points, four or more carbon branch points, or five or more carbon branch points, that are each independently directly bonded to three or more groups independently selected from alkyl, alkoxy, alkenoxy, heteroalkyl, and heteroalkenyl groups, each independently having five or more members, and each optionally oxo-substituted and optionally halogen-substituted.
  • the permanently charged cationic lipid in these cases can include additional carbon branch points, so long as at least two of the carbon branch points are each independently directly bonded to three or more groups independently selected from alkyl, alkenyl, alkoxy, alkenoxy, heteroalkyl, and heteroalkenyl groups, each independently having five or more members, and each optionally oxo-substituted and optionally halogen-substituted.
  • two or more carbon branch points of the permanently charged cationic lipid of the provided lipid nanoparticle are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkoxy, alkenoxy, or heteroalkyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkoxy, alkenoxy, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkoxy, alkenoxy, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly directly
  • 26 78645288V.1 bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenyl, alkoxy, alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points of the permanently charged cationic lipid of the provided lipid nanoparticle are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkoxy, or alkenoxy, groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkoxy, or heteroalkyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkoxy, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkenoxy, or heteroalkyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkenoxy, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy,
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, alkenoxy, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkenoxy, or heteroalkyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkenoxy, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, alkenoxy, or heteroalkyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, alkenoxy, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenyl, alkoxy, alkenoxy, or heteroalkyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenyl, alkoxy, alkenoxy, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenyl, alkoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenyl, alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkoxy, alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points of the permanently charged cationic lipid of the provided lipid nanoparticle are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, or alkoxy groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo- substituted and optionally halogen-substituted alkyl, alkenyl, or alkenoxy groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, or heteroalkyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, or alkenoxy groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, or heteroalkyl groups, each independently having five or more members. In some embodiments, two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, or heteroalkenyl groups, each independently having five or more members. In some embodiments, two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenoxy or
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenoxy, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo- substituted and optionally halogen-substituted alkyl, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenyl, alkoxy, or alkenoxy groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenyl, alkoxy, or heteroalkyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenyl, alkoxy, or heteroalkenyl groups, each independently having five or more members. In some embodiments, two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenyl, alkoxy, or heteroalkyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenyl, alkenoxy, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkoxy, alkenoxy, or heteroalkyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkoxy, alkenoxy, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points of the permanently charged cationic lipid of the provided lipid nanoparticle are each independently directly bonded to three
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl or alkoxy groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl or alkenoxy groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl or heteroalkyl groups, each independently having five or more members. In some embodiments, two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl or heteroalkenyl groups, each independently having five or more members. In some embodiments, two or more carbon branch points are each independently directly bonded to three or more optionally oxo- substituted and optionally halogen-substituted alkenyl or alkoxy groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenyl or alkenoxy groups, each independently having five or more members. In some embodiments, two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenyl or heteroalkyl groups, each independently having five or more members. In some embodiments, two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenyl or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkoxy or alkenoxy groups, each independently having five or more members. In some embodiments, two or more carbon branch points are each independently directly bonded to three or more optionally oxo- substituted and optionally halogen-substituted alkoxy or heteroalkyl groups, each independently having five or more members. In some embodiments, two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkoxy or heteroalkenyl groups, each independently having five or more members. In some embodiments, two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenoxy or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted heteroalkyl or heteroalkenyl groups, each independently having five or more members.
  • two or more carbon branch points of the permanently charged cationic lipid of the provided nanoparticle are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkoxy, alkenoxy groups, each independently having five or more members. In some embodiments, two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkenoxy groups, each independently having five or more members. In some embodiments, two or more carbon branch points are each independently directly bonded to three or more optionally oxo- substituted and optionally halogen-substituted heteroalkyl groups, each independently having five or more members.
  • two or more carbon branch points are each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted heteroalkenyl groups, each independently having five or more members.
  • at least one carbon branch point of the permanently charged cationic lipid of the provided lipid nanoparticle is directly bonded to at least two optionally oxo-substituted and optionally halogen-substituted alkyl groups, each independently having five or more members.
  • At least one carbon branch point is directly bonded to at least two optionally oxo-substituted and optionally halogen-substituted alkenyl groups, each independently having five or more members. In some embodiments, at least one carbon branch point is directly bonded to at least two optionally oxo-substituted and optionally halogen-substituted alkoxy groups, each independently having five or more members. In some embodiments, at least one carbon branch point is directly bonded to at least two optionally oxo-
  • At least one carbon branch point is directly bonded to at least two optionally oxo-substituted and optionally halogen-substituted heteroalkyl groups, each independently having five or more members. In some embodiments, at least one carbon branch point is directly bonded to at least two optionally oxo-substituted and optionally halogen-substituted heteroalkenyl groups, each independently having five or more members.
  • the permanently charged cationic lipid of the provided lipid nanoparticle can further include three or more terminal hydrocarbon chains not directly bonded to the one or more quaternary ammonium nitrogens, where the terminal hydrocarbon chains are each independently alkyl or alkenyl.
  • the permanently charged cationic lipid includes at least one terminal alkyl group not directly bonded to a quaternary ammonium nitrogen.
  • the permanently charged cationic lipid includes at least two terminal alkyl group not directly bonded to a quaternary ammonium nitrogen.
  • the permanently charged cationic lipid includes at least three terminal alkyl group not directly bonded to a quaternary ammonium nitrogen. In some embodiments, the permanently charged cationic lipid includes at least one terminal alkenyl group not directly bonded to a quaternary ammonium nitrogen. In some embodiments, the permanently charged cationic lipid includes at least two terminal alkenyl group not directly bonded to a quaternary ammonium nitrogen. In some embodiments, the permanently charged cationic lipid includes at least three terminal alkenyl group not directly bonded to a quaternary ammonium nitrogen. [0094] Additionally or alternatively, the permanently charged cationic lipid of the provided lipid nanoparticle can include one or more hydrolyzable linkages.
  • the hydrolyzable linkages of the permanently charged cationic lipid can provide the lipid with desirable biodegradability properties, balancing the need for a lipid nanoparticle to have high enough stability to protect nucleic acid cargo for delivery to a targeted cell, and low enough stability to avoid toxicity and off-target effects that can be associated with treatment compounds that insufficiently degrade in a physiological environment.
  • lipid nanoparticles including the permanently charged cationic lipid exhibit the advantageous properties described herein when the hydrolyzable linkages of the permanently charged cationic lipid do not include an ester linkage having the structure C(O)OC.
  • permanently charged cationic lipid include or consist of silyl ether linkages.
  • the hydrolyzable linkages of the permanently charged cationic lipid include or consist of siloxane linkages.
  • the hydrolyzable linkages of the permanently charged cationic lipid include or consist of disulfide linkages.
  • the hydrolyzable linkages of the permanently charged cationic lipid include or consist of carbonate linkages.
  • the hydrolyzable linkages of the permanently charged cationic lipid include or consist of pyrrolidine dicarboxylate linkages.
  • the hydrolyzable linkages of the permanently charged cationic lipid include or consist of cyclic benzylidene linkages.
  • the amount of permanently charged cationic lipid included in the provided lipid nanoparticle has also been shown to provide the particle with its advantageous characteristics.
  • the permanently charged cationic lipid or the salt thereof comprises between about 0.5 mol% and about 30 mol% of the total lipid of the lipid nanoparticle, e.g., between about 0.5 mol% and about 13 mol%, between about 0.5 mol% and about 5.8 mol%, between about 0.5 mol% and about 2.6 mol%, between about 0.5 mol% and about 1.1 mol%, between about 1.1 mol% and about 30 mol%, between about 1.1 mol% and about 13 mol%, between about 1.1 mol% and about 5.8 mol%, between about 1.1 mol% and about 2.6 mol%, between about 2.6 mol% and about 30 mol%, between about 2.6 mol% and about 13 mol%, between about 2.6 mol% and about 5.8 mol%, between about 5.8 mol% and about 30 mol%, between about 5.8 mol% and about 30 mol%, between about 5.8 mol% and about 30 mol%, between about 5.8 mol%
  • the provided lipid nanoparticle generally includes at least one ionizable lipid or a pharmaceutically acceptable salt thereof.
  • the ionizable lipid can be selected or designed to provide the lipid nanoparticle with a desired particle size, stability, and capacity for encapsulation of nucleic acids. Further, lipid nanoparticles including the particular ionizable lipids described herein exhibit, for example, enhanced targeting, optimized clearance rates, and decreased toxicity and immune stimulation. In some embodiments, the lipid nanoparticle includes one species of ionizable lipid.
  • the lipid nanoparticle includes two or more species of ionizable lipids, e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more species of ionizable lipids.
  • Ionizable lipids which can be used with the provided lipid include, for example, N,N- dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N- dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl- N,N-dimethylammonium bromide (DDAB), N,N-(1-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP), 3 -(N-(N′,N′-dimethylaminoethane)- carbamoyl)cholesterol (DC-DODAC), 1,2-dioleyloxy-
  • the permanently charged cationic lipid includes one or more quaternary ammonium nitrogens and the ionizable lipid is nearly structurally identical to the permanently charged cationic lipid of the lipid nanoparticle, where the only structural difference between the ionizable lipid and the permanently charged cationic lipid is that the ionizable lipid has a tertiary nitrogen at the location of each quaternary ammonium nitrogen of the permanently charged cationic lipid.
  • the ionizable lipid of the provided lipid nanoparticle includes a silicon-containing lipid as described and exemplified in International Patent Application Publication No. WO 2020/097520.
  • the ionizable lipid can include or consist of N,N-dimethyl-3-(tris(decan-3-yloxy)silyl)propan-1-amine, N,N-dimethyl-3- (tris(((Z)-dec-4-en-1-yl)oxy)silyl)propan-1-amine, 1-(3-(tris(((Z)-dec-4-en-1- yl)oxy)silyl)propyl)piperidine, N,N-dimethyl-4-(tris(((Z)-dec-4-en-1-yl)oxy)silyl)butan-1- amine, 1-(4-(tris(((Z)-dec-4-en-1-yl)oxy)silyl)butyl)piperidine, N,N-dimethyl-3- (tris(nonyloxy)silyl)propan-1-amine, N,N-diethyl-4-(tris(((Z)-dec-4-)prop
  • the ionizable lipid of the provided lipid nanoparticle includes a silicon-containing ionizable lipid as described and/or exemplified in International Patent Application Publication No. WO 2021/163339, a pyrrolidine-containing ionizable lipid as described and/or exemplified in Internation Patent Application Publication No. WO 2025/052278, and/or an ionizable lipid as
  • the ionizable lipid present in the provided lipid nanoparticle can comprise, for example, between about 30 mol% and about 70 mol% of the total lipid present in the particle, e.g., between about 30 mol% and about 62 mol%, between about 30 mol% and about 54 mol%, between about 30 mol% and about 46 mol%, between about 30 mol% and about 38 mol%, between about 38 mol% and about 70 mol%, between about 38 mol% and about 62 mol%, between about 38 mol% and about 54 mol%, between about 38 mol% and about 46 mol%, between about 46 mol% and about 70 mol%, between about 46 mol% and about 62 mol%, between about 46 mol% and about 54 mol%, between about 54 mol% and about 70 mol%, between about 54 mol% and about 70 mol%, between about 54 mol% and about mol%, between about 54 mol% and about 70 mol%, between about 54 mol%
  • lipid nanoparticle not only the absolute amount of ionizable lipid in a lipid nanoparticle, but also the relative amount of the ionizable lipid relative to that of the permanently charged cationic lipid of the lipid nanoparticle, can enhance the properties of the lipid nanoparticle.
  • the molar ratio of the ionizable lipid or the pharmaceutically acceptable salt thereof in the lipid nanoparticle to the permanently charged cationic lipid of the pharmaceutically acceptable salt thereof in the lipid nanoparticle can be between about 2:1 and about 60:1, e.g., between about 2:1 and about 30:1, between about 2:1 and about 15:1, between about 2:1 and about 7.8:1, between about 2:1 and about 3.9:1, between about 3.9:1 and about 60:1, between about 3.9:1 and about 30:1, between about 3.9:1 and about 15:1, between about 3.9:1 and about 7.8:1, between about 7.8:1 and about 60:1, between about 7.8:1 and about 30:1, between about 7.8:1 and about 15:1, between about 15:1 and about 60:1, between about 15:1 and about 30:1, or between about 30:1 and about 60:1.
  • the lipid nanoparticles disclosed herein generally include at least one phospholipid or a pharmaceutically acceptable salt thereof.
  • the phospholipid can be selected or designed to provide the lipid nanoparticle with improved performance in the delivery of a nucleic acid, e.g., enhanced targeting of hepatic stellate cells, decreased toxicity, good biodegradability, and high biocompatibility.
  • the lipid nanoparticle includes one species of
  • the lipid nanoparticle includes two or more species of phospholipids, e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more species of phospholipids.
  • the phospholipid of the lipid nanoparticle disclosed herein includes or consists of a phosphatidylcholine.
  • Phosphatidylcholine lipids suitable for use with the provided lipid nanoparticles include, for example, 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-didecanoyl-sn-glycero-3-phosphocholine, 1,2-diundecanoyl-sn- glycero-3-phosphocholine, 1,3-dipalmitoyl-rac-glycero-2-phosphocholine, 1,2-dilauroyl-sn- glycero-3-phosphocholine (DLPC), 1,2-ditridecanoyl-sn-glycero-3-phosphocholine, 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipentadecanoyl-sn-glycero-3- phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dihept
  • the phospholipid of the lipid nanoparticle disclosed herein includes or consists of a phosphatidylethanolamine.
  • Phosphatidylethanolamine lipids suitable for use with the provided lipid nanoparticles include, for example, 1,2-diheptadecanoyl-sn- glycero-3-phosphoethanolamine, 1,2-didecanoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dipentadecanoyl-sn-glycero-3- phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), 1,2-dipalmito
  • the phospholipid of the lipid nanoparticle disclosed herein includes or consists of a phosphatidylserine.
  • Phosphatidylserine lipids suitable for use with the provided lipid nanoparticles include, for example, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diheptadecanoyl-sn-glycero-3-phospho-L-serine, 1,2-distearoyl-sn-glycero-3- phospho-L-serine (DSPS), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1,2- didecanoyl-sn-glycero-3-phospho-L-serine, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1,2-dilauroyl-sn
  • DOPS 1,2-
  • the phospholipid of the lipid nanoparticle disclosed herein includes or consists of a phosphatidylglycerol.
  • Phosphatidylglycerol lipids suitable for use with the provided lipid nanoparticles include, for example, 1,2-dioleoyl-sn-glycero-3-phospho-(1'- rac-glycerol) (DOPG), 1,2-diheptadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2- didecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dilauroyl-sn-glycero-3-phospho-(1'- rac-glycerol) (DLPG), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), 1,2- dipentadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dipalmitoyl-sn-glycero
  • the amount of phospholipid included in the provided lipid nanoparticle has also been shown to provide the particle with its advantageous characteristics.
  • the phospholipid present in the provided lipid nanoparticle can comprise, for example, between about 1 mol% and about 20 mol% of the total lipid present in the particle, e.g., between about 1 mol% and about 16.2 mol%, between about 1 mol% and about 12.4 mol%, between about 1 mol% and about 8.6 mol%, between about 1 mol% and about 4.8 mol%, between about 4.8 mol% and about 20 mol%, between about 4.8 mol% and about 16.2 mol%, between about 4.8 mol% and about 12.4 mol%, between about 4.8 mol% and about 8.6 mol% of the total lipid present in the particle, between about 8.6 mol% and about 20 mol%, between about 8.6 mol% and about 16.2 mol%, between about 8.6 mol% and about 12.4 mol
  • the lipid nanoparticles disclosed herein generally include a sterol, e.g., cholesterol, or one or more derivatives thereof.
  • the sterol can be selected or designed to provide the lipid nanoparticle, or a vaccine that includes the lipid nanoparticle, with improved performance in the delivery of a nucleic acid, e.g., enhanced targeting of hepatic stellate cells, decreased toxicity, good biodegradability, and high biocompatibility.
  • the lipid nanoparticle includes one species of sterol.
  • the lipid nanoparticle includes two or more species of sterols, e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more species of sterols.
  • the lipid nanoparticle can include, for example, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether, cholesterol hydroxyethyl ether, cholesterol hydroxyhexyl ether, cholesterol stearate, cholesterol oleate, 7- betahydroxycholesterol, 7-alphahydroxycholesterol, 4-betahydroxycholesterol, cholesterol PEG, beta sitosterol, or any combination thereof.
  • the lipid nanoparticle includes cholesterol, but substantially no derivative of cholesterol.
  • the amount of cholesterol included in the provided lipid nanoparticle has also been shown to provide the lipid nanoparticle with its advantageous characteristics.
  • the cholesterol comprises between about 20 mol% and about 60 mol% of the total lipid of the lipid nanoparticle, e.g., between about 20 mol% and about 52 mol%, between about 20 mol% and about 44 mol%, and between about 20 mol% and about 36 mol%, between about 20 mol% and about 28 mol%, between about 28 mol% and about 60 mol%, between about 28 mol% and about 44 mol%, between about 28 mol% and about 36 mol%, between about 36 mol% and about 60 mol%, between about 36 mol% and about 52 mol%, between about 36 mol% and about 44 mol%, between about 44 mol% and about 60 mol%, between about 44 mol% and about 52 mol%, or between about 52 mol% and about 60 mol% of the total lipid
  • the lipid nanoparticle includes one species of conjugated lipid. In other embodiments, the lipid nanoparticle includes two or more species of conjugated lipids, e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more species of conjugated lipids.
  • the one or more conjugated lipids can each independently be selected or configured to control the size of the particle during formation, and/or to prevent particle aggregation by sterically stabilizing the lipid nanoparticle.
  • the conjugated lipid of the lipid nanoparticle can be situated at the surface of the particle, with the hydrophilic polymer of the conjugated lipid oriented outwardly and interfacing with the aqueous environment, and the lipid component of the conjugated lipid buried in the particle to anchor it in place.
  • Conjugated lipids suitable for use with the provided lipid nanoparticle include, but are not limited to, polyethylene glycol (PEG)-lipid conjugates, ATTA-lipid conjugates, cationic-polymer-lipid conjugates (CPLs), polyoxazoline (POZ)-lipids, polysarcosine (pSAR)- lipids and mixtures thereof.
  • the lipid nanoparticle comprises either a PEG-lipid conjugate or an ATTA-lipid conjugate optionally together with a CPL.
  • the conjugated lipid includes or consists of a PEG-lipid.
  • PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g., International Patent Application Publication No. WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in, e.g., U.S. Patent Application Publication Nos.2003/0077829 and 2005/0008689, PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides as described in, e.g., U.S. Pat. No.
  • PEG-DAA dialkyloxypropyls
  • PEG-DAG diacylglycerol
  • PEG-PE PEG coupled to phospholipids
  • PEG conjugated to ceramides as described in, e.g., U.S. Pat. No.
  • PEG conjugated to cholesterol or a derivative thereof mPEG(2000)-N,N-ditetradecylacetamide (ALC-0159), mPEG(2000)-stearate, 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000), R-3-[( ⁇ - methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine (PEG-C- DOMG), any of the PEG lipids described in International Patent Application Publication No. WO 2022/133344, any of the PEG lipids described in International Patent Application Publication No.
  • the provided lipid nanoparticle can include one or more methyl capped PEG-lipids, one or more uncapped PEG-lipids, or a combination thereof.
  • PEG is a linear, water-soluble polymer of ethylene glycol repeating units. PEGs are classified by their molecular weights; for example, PEG 2000 has a number average molecular weight of about 2,000 daltons, and PEG 5000 has a number average molecular weight of about 5,000 daltons. Such molecular weights are average molecular weights due to polydispersity. PEGs are commercially available from Sigma Chemical Co.
  • MePEG-OH monomethoxypolyethylene glycol
  • MePEG-S monomethoxypolyethylene glycol-succinate
  • MePEG-NHS monomethoxypolyethylene glycol succinimidyl succinate
  • MePEG-NH2 monomethoxypolyethylene glycol-amine
  • MePEG-TRES monomethoxypolyethylene glycol-tresylate
  • MePEG-IM monomethoxypolyethylene glycol-imidazolyl-carbonyl
  • Other PEGs such as those described in U.S. Pat. Nos.
  • 6,774,180 and 7,053,150 are also useful for preparing the PEG-lipid conjugates of the present disclosure.
  • the disclosures of these patents are herein incorporated by reference in their entirety for all purposes.
  • monomethoxypolyethylene glycol acetic acid (MePEG-CH 2 COOH) is particularly useful for preparing PEG-lipid conjugates including, e.g., PEG-DAA conjugates.
  • the PEG moiety of the PEG-lipid conjugates described herein may have a number average molecular weight ranging from about 550 daltons to about 10,000 daltons.
  • the PEG moiety has a number average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG moiety has a number average molecular weight of about 2,000 daltons or about 750 daltons. [0115] In certain instances, the PEG can be optionally substituted by an alkyl, alkoxy, acyl, or aryl group.
  • the PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety.
  • Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties.
  • the linker moiety is a non-ester containing linker moiety.
  • non-ester containing linker moiety refers to a linker moiety that does not contain a carboxylic ester bond (-OC(O)-).
  • Suitable non-ester containing linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (NHC(O)O-), urea (-NHC(O)NH-), disulphide (-S-S-), ether (-O-), succinyl ((O)CCH2CH2C(O)-), succinimidyl (-NHC(O)CH2CH2C(O)NH-), as well as combinations
  • a linker containing both a carbamate linker moiety and an amido linker moiety such as a linker containing both a carbamate linker moiety and an amido linker moiety.
  • a carbamate linker is used to couple the PEG to the lipid.
  • an ester containing linker moiety is used to couple the PEG to the lipid. Suitable ester containing linker moieties include, e.g., carbonate (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof.
  • hydrophilic polymers can be used in place of PEG.
  • suitable polymers include, but are not limited to, ATTA, cationic-polymers, polyoxazoline (POZ), polysarcosine, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, poly (ethyl ethylene phosphate) (PEEP), and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
  • POZ polyoxazoline
  • PEEP poly (ethyl ethylene phosphate)
  • derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
  • the conjugated lipid of the provided lipid nanoparticles includes or consists of a polyoxazoline conjugated lipid, e.g., any of those described in International Patent Application Publication No. WO 2023/144792.
  • the conjugated lipid of the provided lipid nanoparticles can include or consist of, e.g., one or more of the following: a polyethylene glycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, or mixtures thereof.
  • the nucleic acid- lipid particles comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate.
  • the conjugated lipids may comprise a PEG-lipid including, e.g., a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or mixtures thereof.
  • the PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), a PEG- dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), a PEG-distearyloxypropyl (C18), or mixtures thereof.
  • PEG-lipid conjugates suitable for use in the provided lipid nanoparticle include, but are not limited to, mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C- DOMG).
  • PEG-C- DOMG mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride
  • the synthesis of PEG-C-DOMG is described in PCT Application No. PCT/US08/88676, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • PEG-lipid conjugates suitable for use in the disclosure include, without limitation, 1-[8′-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3′,6′- dioxaoctanyl]carbamoyl-w-methylpoly(ethylene glycol) (2 KPEG-DMG). The synthesis of 2
  • the conjugated lipid comprises between about 0.1 mol% and about 5 mol% of the total lipid of the lipid nanoparticle, e.g., between about 0.1 mol% and about 4 mol%, between about 0.1 mol% and about 3 mol%, and between about 0.1 mol% and about 2 mol%, between about 0.1 mol% and about 1 mol%, between about 1 mol% and about 5 mol%, between about 1 mol% and about 4 mol%, between about 1 mol% and about 3 mol%, between about 1 mol% and about 2 mol%, between about 2 mol% and about 5 mol%, between about 2 mol% and about 4 mol%, between about 2 mol% and about 3 mol%, between about 3 mol% and about 5 mol%, between about 3 mol% and about 4 mol%, or between about 4 mol% and about 5 mol% of the total lipid of the lipid nanoparticle.
  • the rate at which the conjugated lipid exchanges out of the lipid nanoparticle can be controlled, for example, by varying the concentration of the lipid conjugate, by varying the molecular weight of the PEG, or by varying the chain length and degree of saturation of the acyl chain groups on the phosphatidylethanolamine or the ceramide.
  • the lipid nanoparticles disclosed herein generally include a nucleic acid.
  • the composition of the lipid nanoparticle can be selected or configured such the nucleic acids present in lipid nanoparticles are resistant in aqueous solution to degradation with a nuclease.
  • the nucleic acid is at least 50% encapsulated within the lipid nanoparticle; in some embodiments, the nucleic acid is at least 75% encapsulated within the lipid nanoparticle; in some embodiments, the nucleic acid is at least 90% encapsulated within
  • the present disclosure provides a lipid nanoparticle formulation comprising a plurality or population of lipid nanoparticles.
  • the nucleic acid is fully encapsulated within the lipid portion of the lipid nanoparticles such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (
  • the nucleic acid that is present in the lipid nanoparticles described herein can include or consist of any form of nucleic acid that is known.
  • the nucleic acids used herein can be single-stranded DNA or RNA (e.g., ssDNA or ssRNA), or double-stranded DNA or RNA (e.g., dsDNA or dsRNA), or DNA-RNA hybrids.
  • Single-stranded nucleic acids include, e.g., mRNA, guide RNA (gRNA), antisense oligonucleotides, ribozymes, mature miRNA, self-amplifying RNA (SAM), and triplex-forming oligonucleotides.
  • double-stranded DNA examples include, e.g., structural genes, genes including control and termination regions, and self- replicating systems such as viral or plasmid DNA.
  • double-stranded RNA examples include, e.g., siRNA and other RNAi agents such as aiRNA and pre-miRNA.
  • Nucleic acids may be of various lengths, generally dependent upon the particular form of nucleic acid.
  • mRNA, plasmids, or genes may be from about 1,000 to about 100,000 nucleotides in length.
  • oligonucleotides may range from about 10 to about 100 nucleotides in length.
  • oligonucleotides, both single-stranded, double-stranded, and triple-stranded may range in length from about 10 to about 60 nucleotides, from about 15 to about 60
  • the nucleic acid of a provided lipid nanoparticle comprises or consists of a modified or substituted polynucleotide or oligonucleotide. Modified or substituted polynucleotides and oligonucleotides can be preferred over native forms in some instances because of properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.
  • the nucleic acid is an RNA molecule comprising at least one modified nucleotide.
  • the RNA molecule comprises one, two, three, four, five, six, seven, eight, nine, ten, or more modified nucleotides in the double- stranded region.
  • the RNA molecule e.g., siRNA
  • less than about 25% (e.g., less than about 25%, 20%, 15%, 10%, or 5%) or from about 1% to about 25% (e.g., from about 1%-25%, 5%-25%, 10%-25%, 15%-25%, 20%-25%, or 10%-20%) of the nucleotides in the double-stranded region comprise modified nucleotides.
  • the RNA molecule comprises modified nucleotides including, but not limited to, 2′-O-methyl (2′Ome) nucleotides, 2′-deoxy-2′-fluoro (2′F) nucleotides, 2′- deoxy nucleotides, 2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof.
  • 2′-O-methyl (2′Ome) nucleotides 2′-deoxy-2′-fluoro (2′F) nucleotides
  • 2′- deoxy nucleotides 2′-O-(2-methoxyethyl) (MOE) nucleotides
  • LNA locked nucleic acid
  • the RNA comprises 2′Ome nucleotides (e.g., 2′Ome purine and/or pyrimidine nucleotides) such as, for example, 2′Ome- guanosine nucleotides, 2′Ome-uridine nucleotides, 2′Ome-adenosine nucleotides, 2′Ome- cytosine nucleotides, and mixtures thereof.
  • the RNA does not comprise 2′Ome-cytosine nucleotides.
  • the RNA comprises a hairpin loop structure.
  • the RNA may comprise modified nucleotides in one strand (i.e., sense or antisense) or both strands of a double-stranded region of the RNA molecule.
  • uridine and/or guanosine nucleotides are modified at selective positions in the double-stranded region of the RNA duplex.
  • at least one, two, three, four, five, six, or more of the uridine nucleotides in the sense and/or antisense strand can be a modified uridine nucleotide such as a 2′Ome-uridine nucleotide.
  • every other nucleotide such as a 2′Ome-uridine nucleotide.
  • uridine nucleotide in the sense and/or antisense strand is a 2′Ome-uridine nucleotide.
  • at least one, two, three, four, five, six, or more of the guanosine nucleotides in the sense and/or antisense strand can be a modified guanosine nucleotide such as a 2′Ome-guanosine nucleotide.
  • every guanosine nucleotide in the sense and/or antisense strand is a 2′Ome-guanosine nucleotide.
  • At least one, two, three, four, five, six, seven, or more 5′-GU- 3′ motifs in an RNA sequence may be modified, e.g., by introducing mismatches to eliminate the 5′-GU-3′ motifs and/or by introducing modified nucleotides such as 2′Ome nucleotides.
  • the 5′-GU-3′ motif can be in the sense strand, the antisense strand, or both strands of the RNA sequence.
  • the 5′-GU-3′ motifs may be adjacent to each other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides.
  • the nucleic acid of a provided lipid nanoparticle includes or consists of an oligonucleotide.
  • Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleotides.
  • a deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose joined covalently to phosphate at the 5′ and 3′ carbons of this sugar to form an alternating, unbranched polymer.
  • a ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose.
  • an oligonucleotide (or a strand thereof) specifically hybridizes to or is complementary to a target polynucleotide sequence.
  • the terms “specifically hybridizable” and “complementary” as used herein indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable.
  • an oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target sequence interferes with the normal function of the target sequence to cause a loss of utility or expression therefrom, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or, in the case of in vitro assays, under conditions in which the assays are conducted.
  • the oligonucleotide may include 1, 2, 3, or more base substitutions as compared to the region of a gene or mRNA sequence that it is targeting or to which it specifically hybridizes.
  • the nucleic acid of a provided lipid nanoparticle is configured or selected to downregulate, e.g., reduce or eliminate, expression of a gene in a cell, e.g., a cell targeted by the lipid nanoparticle.
  • the nucleic acid can be configured or selected to reduce or eliminate expression of a gene in a hepatic stellate cell.
  • the nucleic acid capable of downregulating gene expression includes or consists of small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), antisense RNA (asRNA), Dicer-substrate small interfering RNA (DsiRNA), Piwi-interacting RNA (piRNA), an antisense oligonucleotide, a morpholino oligonucleotide, or a combination thereof.
  • siRNA small interfering RNA
  • miRNA microRNA
  • shRNA short hairpin RNA
  • asRNA antisense RNA
  • DsiRNA Dicer-substrate small interfering RNA
  • piRNA Piwi-interacting RNA
  • an antisense oligonucleotide a morpholino oligonucleotide, or a combination thereof.
  • siRNA small interfering RNA
  • miRNA microRNA
  • shRNA short hairpin RNA
  • asRNA anti
  • the siRNA may be of about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, and is preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably about 18-22, 19-20, or 19-21 base pairs in length).
  • siRNA duplexes may include 3′ overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and 5′ phosphate termini.
  • Examples of siRNA include, without limitation, a double-stranded polynucleotide molecule assembled from two separate stranded molecules, wherein one strand is the sense strand and the other is the complementary antisense strand; a double-stranded polynucleotide molecule assembled from a single stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; a double- stranded polynucleotide molecule with a hairpin secondary structure having self- complementary sense and antisense regions; and a circular single-stranded polynucleotide molecule with two or more loop structures and a stem having self-complementary sense and antisense regions, where the circular polynucleotide can be processed in
  • the immunostimulatory properties of the modified siRNA molecule and its ability to silence target gene expression can be balanced or optimized by the introduction of minimal and selective 2′Ome modifications within the siRNA sequence such as, e.g., within the double-stranded region of the siRNA duplex.
  • the modified siRNA is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 3545%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% less immunostimulatory than the corresponding unmodified siRNA.
  • the immunostimulatory properties of the modified siRNA molecule and the corresponding unmodified siRNA molecule can be determined by, for example, measuring INF-a and/or IL-6 levels from about two to about twelve hours after systemic administration in a mammal or transfection of a mammalian responder cell using an appropriate lipid-based delivery system (such as the LNP delivery system disclosed herein).
  • a modified siRNA molecule has an IC50 (i.e., half-maximal inhibitory concentration) less than or equal to ten-fold that of the corresponding unmodified siRNA (i.e., the modified siRNA has an IC50 that is less than or equal to ten-times the IC50 of the corresponding unmodified siRNA).
  • the modified siRNA has an IC50 less than or equal to three-fold that of the corresponding unmodified siRNA sequence.
  • the modified siRNA has an IC50 less than or equal to two-fold that of the corresponding unmodified siRNA.
  • a modified siRNA molecule is capable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 20 80%, 85%, 90%, 95%, or 100% of the expression of the target sequence relative to the corresponding unmodified siRNA sequence.
  • the siRNA molecule does not comprise phosphate backbone modifications, e.g., in the sense and/or antisense strand of the double-stranded region.
  • the siRNA comprises one, two, three, four, or more phosphate backbone modifications, e.g., in the sense and/or antisense strand of the double-stranded region.
  • the siRNA does not comprise phosphate backbone modifications.
  • the siRNA does not comprise 2′-deoxy nucleotides, e.g., in the sense and/or antisense strand of the double-stranded region.
  • the siRNA comprises one, two, three, four, or more 2′-deoxy nucleotides, e.g., in the sense and/or antisense strand of the double-stranded region.
  • the siRNA does not comprise 2′-deoxy nucleotides.
  • the nucleotide at the 3′ end of the double-stranded region in the sense and/or antisense strand is not a modified nucleotide.
  • the nucleotides near the 3′ end (e.g., within one, two, three, or four nucleotides of the 3′ end) of the double-stranded region in the sense and/or antisense strand are not modified nucleotides.
  • the siRNA molecules described herein may have 3′ overhangs of one, two, three, four, or more nucleotides on one or both sides of the double-stranded region, or may lack overhangs (i.e., have blunt ends) on one or both sides of the double-stranded region.
  • the siRNA has 3′ overhangs of two nucleotides on each side of the double-stranded region.
  • the 3′ overhang on the antisense strand has complementarity to the target sequence and the 3′ overhang on the sense strand has complementarity to a complementary strand of the target sequence.
  • the 3′ overhangs do not have complementarity to the target sequence or the complementary strand thereof.
  • the 3′ overhangs comprise one, two, three, four, or more nucleotides such as 2′-deoxy (2′H) nucleotides.
  • the 3′ overhangs comprise deoxythymidine (dT) and/or uridine nucleotides.
  • one or more of the nucleotides in the 3′ overhangs on one or both sides of the double-stranded region comprise modified nucleotides.
  • modified nucleotides include 2′OMe nucleotides, 2′-deoxy-2′F nucleotides, 2′-deoxy nucleotides, 2′-O-2-MOE nucleotides, LNA nucleotides, and mixtures thereof.
  • one, two, three, four, or more nucleotides in the 3′ overhangs present on the sense and/or antisense strand of the siRNA comprise 2′Ome nucleotides (e.g., 2′Ome purine and/or pyrimidine nucleotides) such as, for example, 2′Ome-guanosine nucleotides, 2′Ome-uridine nucleotides, 2′Ome-adenosine nucleotides, 2′Ome-cytosine nucleotides, and mixtures thereof.
  • 2′Ome nucleotides e.g., 2′Ome purine and/or pyrimidine nucleotides
  • 2′Ome-guanosine nucleotides e.g., 2′Ome-uridine nucleotides
  • 2′Ome-adenosine nucleotides e.g., 2′Ome-cytosine nucleotides, and mixtures thereof.
  • the siRNA may comprise at least one or a cocktail (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) of unmodified and/or modified siRNA sequences that silence target gene expression.
  • the cocktail of siRNA may comprise sequences which are directed to the same region or domain (e.g., a “hot spot”) and/or to different regions or domains
  • one or more modified siRNA that silence target gene expression are present in a cocktail.
  • one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) unmodified siRNA sequences that silence target gene expression are present in a cocktail.
  • the antisense strand of the siRNA molecule comprises or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence or a portion thereof. In other embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that is 100% complementary to the target sequence or a portion thereof. In further embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that specifically hybridizes to the target sequence or a portion thereof.
  • the sense strand of the siRNA molecule comprises or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the target sequence or a portion thereof. In additional embodiments, the sense strand of the siRNA molecule comprises or consists of a sequence that is 100% identical to the target sequence or a portion thereof. a) Selection of siRNA Sequences [0144] Suitable siRNA sequences can be identified using any means known in the art.
  • the nucleotides immediately 3′ to the dinucleotide sequences are identified as potential siRNA sequences (i.e., a target sequence or a sense strand sequence).
  • potential siRNA sequences i.e., a target sequence or a sense strand sequence.
  • the 19, 21, 23, 25, 27, 29, 31, 33, 35, or more nucleotides immediately 3′ to the dinucleotide sequences are identified as potential siRNA sequences.
  • the dinucleotide sequence is an AA or NA sequence and the 19 nucleotides immediately 3′ to the AA or NA dinucleotide are identified as potential siRNA sequences.
  • siRNA sequences are usually spaced at different positions along the length of the target gene. To further enhance silencing efficiency of the siRNA sequences, potential siRNA sequences
  • 52 78645288V.1 may be analyzed to identify sites that do not contain regions of homology to other coding sequences, e.g., in the target cell or organism. For example, a suitable siRNA sequence of about 21 base pairs typically will not have more than 16-17 contiguous base pairs of homology to coding sequences in the target cell or organism. If the siRNA sequences are to be expressed from an RNA Pol III promoter, siRNA sequences lacking more than 4 contiguous A’s or T’s are selected. [0145] Once a potential siRNA sequence has been identified, a complementary sequence (i.e., an antisense strand sequence) can be designed. A potential siRNA sequence can also be analyzed using a variety of criteria known in the art.
  • the siRNA sequences may be analyzed by a rational design algorithm to identify sequences that have one or more of the following features: (1) G/C content of about 25% to about 60% G/C; (2) at least 3 A/Us at positions 15-19 of the sense strand; (3) no internal repeats; (4) an A at position 19 of the sense strand; (5) an A at position 3 of the sense strand; (6) a U at position 10 of the sense strand; (7) no G/C at position 19 of the sense strand; and (8) no G at position 13 of the sense strand.
  • sequences with one or more of the foregoing characteristics may be selected for further analysis and testing as potential siRNA sequences.
  • siRNA sequences with one or more of the following criteria can often be eliminated as siRNA: (1) sequences comprising a stretch of 4 or more of the same base in a row; (2) sequences comprising homopolymers of Gs (i.e., to reduce possible non- specific effects due to structural characteristics of these polymers; (3) sequences comprising triple base motifs (e.g., GGG, CCC, AAA, or TTT); (4) sequences comprising stretches of 7 or more G/Cs in a row; and (5) sequences comprising direct repeats of 4 or more bases within the candidates resulting in internal fold-back structures.
  • potential siRNA sequences may be further analyzed based on siRNA duplex asymmetry as described in, e.g., Khvorova et al., Cell, 115:209-216 (2003); and Schwarz et al., Cell, 115: 199-208 (2003).
  • potential siRNA sequences may be further analyzed based on secondary structure at the target site as described in, e.g., Luo et al., Biophys. Res. Commun., 318:303-310 (2004). For example, secondary structure at the target site can be modeled using the Mfold algorithm (available at
  • siRNA sequences which favor accessibility at the target site where less secondary structure in the form of base-pairing and stem-loops is present.
  • the sequence can be analyzed for the presence of any immunostimulatory properties, e.g., using an in vitro cytokine assay or an in vivo animal model.
  • Motifs in the sense and/or antisense strand of the siRNA sequence such as GU-rich motifs (e.g., 5′-GU-3′, 5′-UGU-3′, 5′-GUGU-3′, 5′-UGUGU-3′, etc.) can also provide an indication of whether the sequence may be immunostimulatory.
  • GU-rich motifs e.g., 5′-GU-3′, 5′-UGU-3′, 5′-GUGU-3′, 5′-UGUGU-3′, etc.
  • an siRNA sequence can be contacted with a mammalian responder cell under conditions such that the cell produces a detectable immune response to determine whether the siRNA is an immunostimulatory or a non-immunostimulatory siRNA.
  • the mammalian responder cell may be from a naive mammal (i.e., a mammal that has not previously been in contact with the gene product of the siRNA sequence).
  • the mammalian responder cell may be, e.g., a peripheral blood mononuclear cell (PBMC), a macrophage, and the like.
  • the detectable immune response may comprise production of a cytokine or growth factor such as, e.g., TNF- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IL-6, IL-12, or a combination thereof.
  • siRNA molecule identified as being immunostimulatory can then be modified to decrease its immunostimulatory properties by replacing at least one of the nucleotides on the sense and/or antisense strand with modified nucleotides. For example, less than about 30% (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5%) of the nucleotides in the double-stranded region of the siRNA duplex can be replaced with modified nucleotides such as 2′Ome nucleotides.
  • the modified siRNA can then be contacted with a mammalian responder cell as described above to confirm that its immunostimulatory properties have been reduced or abrogated.
  • Suitable in vitro assays for detecting an immune response include, but are not limited to, the double monoclonal antibody sandwich immunoassay technique of David et al. (U.S. Pat. No.4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970)); the “Western blot” method of Gordon et al. (U.S. Pat. No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., J. Biol.
  • a non-limiting example of an in vivo model for detecting an immune response includes an in vivo mouse cytokine induction assay as described in, e.g., Judge et al., Mol. Ther., 13:494- 505 (2006).
  • the assay that can be performed as follows: (1) siRNA can be administered by standard intravenous injection in the lateral tail vein; (2) blood can be collected by cardiac puncture about 6 hours after administration and processed as plasma for cytokine analysis; and (3) cytokines can be quantified using sandwich ELISA kits according to the manufacturer’s instructions (e.g., mouse and human IFN-a (PBL Biomedical; Piscataway, N.J.); human IL-6 and TNF-a (eBioscience; San Diego, Calif); and mouse IL-6, TNF-a, and IFN-y (BD Biosciences; San Diego, Calif)).
  • sandwich ELISA kits e.g., mouse and human IFN-a (PBL Biomedical; Piscataway, N.J.); human IL-6 and TNF-a (eBioscience; San Diego, Calif); and mouse IL-6, TNF-a, and IFN-y (BD Biosciences; San Diego, Calif)).
  • Monoclonal antibodies that specifically bind cytokines and growth factors are commercially available from multiple sources and can be generated using methods known in the art (see, e.g., Kohler et al., Nature, 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)). Generation of monoclonal antibodies has been previously described and can be accomplished by any means known in the art (Buhring et al., in Hybridoma, Vol.10, No.1, pp. 77-78 (1991)).
  • siRNA can be provided in several forms including, e.g., as one or more isolated small- interfering RNA (siRNA) duplexes, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a transcriptional cassette in a DNA plasmid. siRNA can be chemically synthesized.
  • siRNA small- interfering RNA
  • oligonucleotides that comprise the siRNA molecules can be synthesized using any of a variety of techniques known in the art, such as those described in Usman et al., J. Am. Chem. Soc., 109:7845 (1987); Scaringe et al., Nucl. Acids Res., 18:5433 (1990); Wincott et al., Nucl. Acids Res., 23:2677-2684 (1995); and Wincott et al., Methods Mol. Bio., 74:59 (1997).
  • RNA population can be used to provide long precursor RNAs, or long precursor RNAs that have substantial or complete identity to a selected target sequence can be used to make the siRNA.
  • the RNAs can be isolated from cells or tissue, synthesized, and/or cloned according to methods well known to those of skill in the art.
  • the RNA can be a mixed population (obtained from cells or tissue, transcribed from cDNA, subtracted, selected, etc.), or can represent a single target sequence.
  • RNA can be naturally occurring (e.g., isolated from tissue or cell samples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCR products or a cloned cDNA), or chemically synthesized.
  • the complement is also transcribed in vitro and hybridized to form a dsRNA. If a naturally occurring RNA population is used, the RNA complements are also provided (e.g., to form dsRNA for digestion by E.coli RNAse III or Dicer), e.g., by transcribing cDNAs corresponding to the RNA population, or by using RNA polymerases.
  • RNAs are then hybridized to form double stranded RNAs for digestion.
  • the dsRNAs can be directly administered to a subject or can be digested in vitro prior to administration.
  • Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see, U.S. Pat. Nos.
  • siRNA are chemically synthesized.
  • siRNA can also be generated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25 nucleotides in length) with the E.coli RNase III or Dicer. These enzymes process the dsRNA into biologically active siRNA (see, e.g., Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci.
  • dsRNA are at least 50
  • dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotides in length, or longer.
  • the dsRNA can encode for an entire gene transcript or a partial gene transcript.
  • siRNA may be encoded by a plasmid (e.g., transcribed as sequences that automatically fold into duplexes with hairpin loops).
  • siRNA molecules can also be synthesized via a tandem synthesis technique, wherein both strands are synthesized as a single continuous oligonucleotide fragment or strand separated 20 by a cleavable linker that is subsequently cleaved to provide separate fragments or strands that hybridize to form the siRNA duplex.
  • the linker can be a polynucleotide linker or a non-nucleotide linker.
  • the tandem synthesis of siRNA can be readily adapted to both multiwell/multiplate synthesis platforms as well as large scale synthesis platforms employing batch reactors, synthesis columns, and the like.
  • siRNA molecules can be assembled from two distinct oligonucleotides, wherein one oligonucleotide comprises the sense strand and the other comprises the antisense strand of the siRNA.
  • each strand can be synthesized separately and joined together by hybridization or ligation following synthesis and/or deprotection.
  • siRNA molecules can be synthesized as a single continuous oligonucleotide fragment, where the self-complementary sense and antisense regions hybridize to form an siRNA duplex having hairpin secondary structure.
  • siRNA molecules comprise a duplex having two strands and at least one modified nucleotide in the double-stranded region, wherein each strand is about 15 to about 60 nucleotides in length.
  • the modified siRNA is less immunostimulatory than a corresponding unmodified siRNA sequence, but retains the capability of silencing the expression of a target sequence.
  • the degree of chemical modifications introduced into the siRNA molecule strikes a balance between reduction or abrogation of the immunostimulatory properties of the siRNA and retention of RNAi activity.
  • an siRNA molecule that targets a gene of interest can be minimally modified (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5% modified) at selective uridine and/or guanosine nucleotides within the siRNA duplex to eliminate the immune response generated by the siRNA while retaining its capability to silence target gene expression.
  • modified nucleotides include, but are not limited to, ribonucleotides having a 2′-O-methyl (2′Ome), 2′-deoxy-2′-fluoro (2′F), 2′-deoxy, 5-C-methyl, 2′-O-(2- methoxyethyl) (MOE), 4′-thio, 2′-amino, or 2′-C-allyl group.
  • Modified nucleotides having a Northern conformation such as those described in, e.g., Saenger, Principles of Nucleic Acid Structure, Springer-Verlag Ed. (1984), are also suitable for use in siRNA molecules.
  • Such modified nucleotides include, without limitation, locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-O-(2-methoxyethyl) (MOE) nucleotides, 2′-methyl-thio-ethyl nucleotides, 2′-deoxy-2′-fluoro (2′F) nucleotides, 2′-deoxy- 2′-chloro (2′Cl) nucleotides, and 2′-azido nucleotides.
  • LNA locked nucleic acid
  • MOE 2-methoxyethyl
  • MOE 2-methoxyethyl) nucleotides
  • 2′-methyl-thio-ethyl nucleotides 2′-methyl-thio-ethyl nucleotides
  • 2′F 2-methoxy-2′-fluoro
  • a G-clamp nucleotide refers to a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine nucleotide within a duplex (see, e.g., Lin et al., J. Am. Chem. Soc., 120:8531-8532 (1998)).
  • nucleotides having a nucleotide base analog such as, for example, C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3- nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucl. Acids Res., 29:2437-2447 (2001)) can be incorporated into siRNA molecules.
  • siRNA molecules may further comprise one or more chemical modifications such as terminal cap moieties, phosphate backbone modifications, and the like.
  • terminal cap moieties include, without limitation, inverted deoxy abasic residues, glyceryl modifications, 4′,5′-methylene nucleotides, 1-( ⁇ -D-erythrofuranosyl) nucleotides, 4′-thio nucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L- nucleotides, ⁇ -nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3′,4′-seco nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5- dihydroxypentyl nucleotides, 3′-3′-inverted nucleotide moieties, 3′-3′-inverted abasic moieties, 3′-2′-inverted nucleotide moieties, 3′-2′-inverted nucle
  • phosphate backbone modifications include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker et al., Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331- 417 (1995); Mesmaeker et al., Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39 (1994)).
  • the sense and/or antisense strand of the siRNA molecule can further comprise a 3′-terminal overhang having about 1 to about 4 (e.g., 1, 2, 3, or 4) 2′-deoxy ribonucleotides and/or any combination of modified and unmodified nucleotides. Additional examples of modified nucleotides and types of chemical modifications that can be introduced into siRNA molecules are described, e.g., in UK Patent No. GB 2,397,818 B and U.S.
  • the siRNA molecules described herein can optionally comprise one or more non- nucleotides in one or both strands of the siRNA.
  • non-nucleotide refers to any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their activity.
  • chemical modification of the siRNA comprises attaching a conjugate to the siRNA molecule.
  • the conjugate can be attached at the 5′ and/or 3′ end of the sense and/or antisense strand of the siRNA via a covalent attachment such as, e.g., a biodegradable linker.
  • the conjugate can also be attached to the siRNA, e.g., through a carbamate group or other linking group (see, e.g., U.S. Patent Application Publication Nos. 2005/0074771, 2005/0043219, and 20050158727).
  • the conjugate is a molecule that facilitates the delivery of the siRNA into a cell.
  • conjugate molecules suitable for attachment to siRNA include, without limitation, steroids such as cholesterol, glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs and derivatives thereof),
  • 59 78645288V.1 sugars e.g., galactose, galactosamine, N-acetyl galactosamine, glucose, mannose, fructose, fucose, etc.
  • phospholipids e.g., galactose, galactosamine, N-acetyl galactosamine, glucose, mannose, fructose, fucose, etc.
  • phospholipids e.g., phospholipids, peptides, ligands for cellular receptors capable of mediating cellular uptake, and combinations thereof (see, e.g., U.S. Patent Application Publication Nos. 2003/0130186, 2004/0110296, and 2004/0249178; U.S. Patent No. 6,753,423).
  • examples include the lipophilic moiety, vitamin, polymer, peptide, protein, nucleic acid, small molecule, oligosaccharide, carbohydrate cluster, intercalator, minor groove binder, cleaving agent, and cross-linking agent conjugate molecules described in U.S. Patent Application Publication Nos.2005/0119470 and 2005/0107325.
  • Yet other examples include the 2′-O-alkyl amine, 2′-O-alkoxyalkyl amine, polyamine, C5-cationic modified pyrimidine, cationic peptide, guanidinium group, amidinium group, and cationic amino acid conjugate molecules described in U.S. Patent Application Publication No. 2005/0153337.
  • Additional examples include the hydrophobic group, membrane active compound, cell penetrating compound, cell targeting signal, interaction modifier, and steric stabilizer conjugate molecules described in U.S. Patent Application Publication No.2004/0167090. Further examples include the conjugate molecules described in U.S. Patent Application Publication No. 2005/0239739.
  • the type of conjugate used and the extent of conjugation to the siRNA molecule can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of the siRNA while retaining RNAi activity.
  • one skilled in the art can screen siRNA molecules having various conjugates attached thereto to identify ones having improved properties and full RNAi activity using any of a variety of well-known in vitro cell culture or in vivo animal models. 2.
  • the nucleic acid of a provided lipid nanoparticle includes or consists of miRNA.
  • miRNA are single-stranded RNA molecules of about 21-23 nucleotides in length which regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed, but miRNAs are not translated into protein (non-coding RNA); instead, each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre-miRNA and finally into a functional mature miRNA. Mature miRNA molecules are either partially or completely complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression.
  • mRNA messenger RNA
  • miRNA molecules The identification of miRNA molecules is described, e.g., in Lagos-Quintana et al., Science, 294:853-858; Lau et al., Science, 294:858-862; and Lee et al., Science, 294:862-864.
  • miRNA 60 78645288V.1
  • the genes encoding miRNA are much longer than the processed mature miRNA molecule. miRNA are first transcribed as primary transcripts or pri-miRNA with a cap and poly-A tail and processed to short, approximately 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. This processing is performed in animals by a protein complex known as the Microprocessor complex, consisting of the nuclease Drosha and the double- stranded RNA binding protein Pasha (Denli et al., Nature, 432:231-235 (2004)).
  • the Microprocessor complex consisting of the nuclease Drosha and the double- stranded RNA binding protein Pasha (Denli et al., Nature, 432:231-235 (2004)).
  • RNA-induced silencing complex (RISC) (Bernstein et al., Nature, 409:363-366 (2001). Either the sense strand or antisense strand of DNA can function as templates to give rise to miRNA.
  • RISC RNA-induced silencing complex
  • This strand is known as the guide strand and is selected by the Argonaute protein, the catalytically active RNase in the RISC complex, on the basis of the stability of the 5′ end (Preall et al., Curr. Biol., 16:530- 535 (2006)).
  • the remaining strand known as the anti-guide or passenger strand, is degraded as a RISC complex substrate (Gregory et al., Cell, 123:631-640 (2005)).
  • miRNAs base pair with their complementary mRNA molecules and induce target mRNA degradation and/or translational silencing.
  • Mammalian miRNA molecules are usually complementary to a site in the 3′ UTR of the target mRNA sequence.
  • the annealing of the miRNA to the target mRNA inhibits protein translation by blocking the protein translation machinery. In certain other instances, the annealing of the miRNA to the target mRNA facilitates the cleavage and degradation of the target mRNA through a process similar to RNA interference (RNAi). miRNA may also target methylation of genomic sites which correspond to targeted mRNA. Generally, miRNA function in association with a complement of proteins collectively termed the miRNP.
  • the miRNA molecules described herein are about 15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotides in length, more typically about 15-30, 15-25, or 19-25 nucleotides in length, and are preferably about 20-24, 21-22, or 21-23 nucleotides in length.
  • miRNA molecules may comprise one or more modified nucleotides.
  • miRNA sequences may comprise one or more of the modified nucleotides described above for siRNA sequences. In a preferred
  • the miRNA molecule comprises 2′OMe nucleotides such as, for example, 2′OMe- guanosine nucleotides, 2′OMe-uridine nucleotides, or mixtures thereof.
  • the nucleic acid of a provided lipid nanoparticle includes or consists of shRNA.
  • An shRNA molecule includes a short RNA sequence that makes a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • the shRNA molecules of the present disclosure may be chemically synthesized or transcribed from a transcriptional cassette in a DNA plasmid.
  • the shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).
  • the shRNA molecules of the invention are typically about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, and are preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double-stranded shRNA is 15-60, 15-50, 15- 40, 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded shRNA is about 15-60, 15-50, 15-40, 15-30, 15- 25, or 19-25 base pairs in length, preferably about 18-22, 19-20, or 19-21 base pairs in length).
  • shRNA duplexes may comprise 3′ overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides on the antisense strand and/or 5′-phosphate termini on the sense strand.
  • the shRNA comprises a sense strand and/or antisense strand sequence of from about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-55, 15-50, 15-45, 15-40, 15-35, 15-30, or 15-25 nucleotides in length), preferably from about 19 to about 40 nucleotides in length (e.g., about 19-40, 19-35, 19-30, or 19-25 nucleotides in length), more preferably from about 19 to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23 nucleotides in length).
  • Non-limiting examples of shRNA include a double-stranded polynucleotide molecule assembled from a single-stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; and a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions.
  • the sense and antisense strands of the shRNA are linked by a loop structure comprising from about 1 to about 25 nucleotides, from about 2 to about 20 nucleotides, from about 4 to about 15 nucleotides, from about 5 to about 12 nucleotides, or 1,
  • shRNA sequences can be identified, synthesized, and modified using any means known in the art for designing, synthesizing, and modifying siRNA sequences. Additional embodiments related to the shRNA molecules of the present disclosure, as well as methods of designing and synthesizing such shRNA molecules, are described in U.S. Patent Application Publication No. 2011/0071208, the disclosure of which is herein incorporated by reference in its entirety for all purposes. 4. asRNA [0173] In some embodiments, the nucleic acid of a provided lipid nanoparticle includes or consists of asRNA.
  • RNA molecules also referred to in the art as antisense transcripts, are naturally-occurring or synthetically produced single-stranded RNA molecules that are complementary to a protein-coding messenger RNA (mRNA) with which it hybridizes and thereby blocks the translation of the mRNA into a protein.
  • Antisense transcript are classified into short (less than 200 nucleotides) and long (greater than 200 nucleotides) non-coding RNAs (ncRNAs).
  • the asRNA molecules may include a sequence complementary to a genomic sequence between 100, 80, 60, 40, 20, or 10 kb upstream of the transcription initiation site of a target gene to downstream of the transcription termination site.
  • the asRNA includes a sequence complementary to a genomic sequence between 1 kb upstream of the transcription initiation site of the target gene and 1 kb downstream of the transcription termination site of the target gene. In other embodiments, the asRNA includes a sequence complementary to a genomic sequence between 500, 250, or 100 nucleotides upstream of the transcription initiation site of the target gene to the downstream 500, 250 or 100 nucleotides of the transcription termination site of the target gene.
  • the asRNA molecule may include a sequence complementary to a genomic sequence comprising the coding region of the target gene.
  • the asRNA may include a sequence complementary to a genomic sequence aligned with a promoter region of the target gene on the template strand.
  • the gene can have a plurality of promoter regions, in this case, the asRNA can be aligned with one, two or more promoter regions.
  • An online database of annotated gene loci can be used to identify a promoter region of a gene.
  • the alignment region between the asRNA molecule and the target gene promoter region may be as short as a single nucleotide in length, or can be at least 15 or at least 20 nucleotide length, or at least 25 nucleotide length, or at least 30, 35, 40, 45, or 50 nucleotide length, or at least 55, 60, 65, 70 or 75 nucleotide length, or at least 100 nucleotide length.
  • the primary natural function of asRNA molecules involves regulating gene expression, and synthetic versions have been used widely as research tools for gene knockdown and for therapeutic applications. asRNA molecules and their functions have been described in the art (see e.g., Weiss et al.
  • the nucleic acid of a provided lipid nanoparticle includes or consists of DsiRNA.
  • DsiRNA molecules are processed in vivo by Dicer to produce an active siRNA which is incorporated into the RISC complex for RNA interference of a target gene.
  • the DsiRNA has a length sufficient such that it is processed by Dicer to produce an siRNA.
  • the DsiRNA comprises (i) a first oligonucleotide sequence (also termed the sense strand) that is between about 25 and about 60 nucleotides in length (e.g., about 25-60, 25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length), preferably between about 25 and about 30 nucleotides in length (e.g., 25, 26, 27, 28, 29, or 30 nucleotides in length), and (ii) a second oligonucleotide sequence (also termed the antisense strand) that anneals to the first sequence under biological conditions, such as the conditions found in the cytoplasm of a cell.
  • a first oligonucleotide sequence also termed the sense strand
  • the second oligonucleotide sequence may be between about 25 and about 60 nucleotides in length (e.g., about 25-60, 25-55, 25-50, 25- 45, 25-40, 25-35, or 25-30 nucleotides in length), and is preferably between about 25 and about 30 nucleotides in length (e.g., 25, 26, 27, 28, 29, or 30 nucleotides in length).
  • a region of one of the sequences, particularly of the antisense strand, of the DsiRNA can have a sequence length of at least about 19 nucleotides, for example, from about 19 to about 60 nucleotides (e.g., about 19-60, 19-55, 19-50, 19-45, 19-40, 19-35, 19-30, or 19-25 nucleotides), preferably from about 19 to about 23 nucleotides (e.g., 19, 20, 21, 22, or 23 nucleotides) that are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to trigger an RNAi response.
  • the DsiRNA has a length sufficient such that it is processed by Dicer to produce an siRNA and has at least one of the following properties: (i) the DsiRNA
  • 64 78645288V.1 is asymmetric, e.g., has a 3′ overhang on the antisense strand; and/or (ii) the DsiRNA has a modified 3′ end on the sense strand to direct orientation of Dicer binding and processing of the DsiRNA to an active siRNA.
  • the sense strand comprises from about 22 to about 28 nucleotides and the antisense strand comprises from about 24 to about 30 nucleotides.
  • the DsiRNA has an overhang on the 3′ end of the antisense strand.
  • the sense strand is modified for Dicer binding and processing by suitable modifiers located at the 3′ end of the sense strand.
  • Suitable modifiers include nucleotides such as deoxyribonucleotides, acyclonucleotides, and the like, and sterically hindered molecules such as fluorescent molecules and the like.
  • nucleotide modifiers When nucleotide modifiers are used, they replace ribonucleotides in the DsiRNA such that the length of the DsiRNA does not change.
  • the DsiRNA has an overhang on the 3′ end of the antisense strand and the sense strand is modified for Dicer processing.
  • the 5′ end of the sense strand has a phosphate.
  • the 5′ end of the antisense strand has a phosphate.
  • the antisense strand or the sense strand or both strands have one or more 2′-O-methyl (2′OMe) modified nucleotides.
  • the antisense strand contains 2′OMe modified nucleotides.
  • the antisense stand contains a 3′ overhang that is comprised of 2′OMe modified nucleotides.
  • the antisense strand could also include additional 2′OMe modified nucleotides.
  • the sense and antisense strands anneal under biological conditions, such as the conditions found in the cytoplasm of a cell.
  • a region of one of the sequences, particularly of the antisense strand, of the DsiRNA can have a sequence length of at least about 19 nucleotides, where these nucleotides are in the 21-nucleotide region adjacent to the 3′ end of the antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene.
  • the DsiRNA may also have one or more of the following additional properties: (a) the antisense strand has a right shift from the typical 21- mer (i.e., the antisense strand includes nucleotides on the right side of the molecule when compared to the typical 21-mer); (b) the strands may not be completely complementary (i.e., the strands may contain simple mismatch pairings); and (c) base modifications such as locked nucleic acid(s) may be included in the 5′ end of the sense strand.
  • the sense strand comprises from about 25 to about 28 nucleotides (e.g., 25, 26, 27, or 28 nucleotides), wherein the 2 nucleotides on the 3′ end of the sense strand are deoxyribonucleotides.
  • the sense strand contains a phosphate at the 5′ end.
  • 65 78645288V.1 antisense strand comprises from about 26 to about 30 nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides) and contains a 3′ overhang of 1-4 nucleotides.
  • the nucleotides comprising the 3′ overhang are modified with 2′OMe modified ribonucleotides.
  • the antisense strand contains alternating 2′OMe modified nucleotides beginning at the first monomer of the antisense strand adjacent to the 3′ overhang, and extending 15-19 nucleotides from the first monomer adjacent to the 3′ overhang.
  • the DsiRNA has a length sufficient such that it is processed by Dicer to produce an siRNA and at least one of the following properties: (i) the DsiRNA is asymmetric, e.g., has a 3′ overhang on the sense strand; and (ii) the DsiRNA has a modified 3′ end on the antisense strand to direct orientation of Dicer binding and processing of the DsiRNA to an active siRNA.
  • the sense strand comprises from about 24 to about 30 nucleotides (e.g., 24, 25, 26, 27, 28, 29, or 30 nucleotides) and the antisense strand comprises from about 22 to about 28 nucleotides (e.g., 22, 23, 24, 25, 26, 27, or 28 nucleotides).
  • the DsiRNA has an overhang on the 3′ end of the sense strand.
  • the antisense strand is modified for Dicer binding and processing by suitable modifiers located at the 3′ end of the antisense strand.
  • Suitable modifiers include nucleotides such as deoxyribonucleotides, acyclonucleotides, and the like, and sterically hindered molecules such as fluorescent molecules and the like.
  • nucleotide modifiers When nucleotide modifiers are used, they replace ribonucleotides in the DsiRNA such that the length of the DsiRNA does not change.
  • the DsiRNA has an overhang on the 3′ end of the sense strand and the antisense strand is modified for Dicer processing.
  • the antisense strand has a 5′ phosphate. The sense and antisense strands anneal under biological conditions, such as the conditions found in the cytoplasm of a cell.
  • a region of one of the sequences, particularly of the antisense strand, of the DsiRNA can have a sequence length of at least 19 nucleotides, wherein these nucleotides are adjacent to the 3′ end of antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene.
  • the DsiRNA may also have one or more of the following additional properties: (a) the antisense strand has a left shift from the typical 21- mer (i.e., the antisense strand includes nucleotides on the left side of the molecule when compared to the typical 21-mer); and (b) the strands may not be completely complementary, i.e., the strands may contain simple mismatch pairings.
  • the DsiRNA has an asymmetric structure, with the sense strand having a 25-base pair length, and the antisense strand having a 27-base pair length with a 2-base 3′ overhang.
  • this DsiRNA having an asymmetric structure further contains 2 deoxynucleotides at the 3′ end of the sense strand in place of two of the ribonucleotides.
  • this DsiRNA having an asymmetric structure further contains 2′OMe modifications at positions 9, 11, 13, 15, 17, 19, 21, 23, and 25 of the antisense strand (wherein the first base at the 5′ end of the antisense strand is position 1).
  • this DsiRNA having an asymmetric structure further contains a 3′ overhang on the antisense strand comprising 1, 2, 3, or 42′OMe nucleotides (e.g., a 3′ overhang of 2′OMe nucleotides at positions 26 and 27 on the antisense strand).
  • DsiRNA may be designed by first selecting an antisense strand siRNA sequence having a length of at least 19 nucleotides.
  • the antisense siRNA is modified to include about 5 to about 11 ribonucleotides on the 5′ end to provide a length of about 24 to about 30 nucleotides.
  • the antisense strand has a length of 21 nucleotides, 3-9, preferably 4-7, or more preferably 6 nucleotides may be added on the 5′ end.
  • the added ribonucleotides may be complementary to the target gene sequence, full complementarity between the target sequence and the antisense siRNA is not required. That is, the resultant antisense siRNA is sufficiently complementary with the target sequence.
  • a sense strand is then produced that has about 22 to about 28 nucleotides.
  • the sense strand is substantially complementary with the antisense strand to anneal to the antisense strand under biological conditions.
  • the sense strand is synthesized to contain a modified 3′ end to direct Dicer processing of the antisense strand.
  • the antisense strand of the dsRNA has a 3′ overhang.
  • the sense strand is synthesized to contain a modified 3′ end for Dicer binding and processing and the antisense strand of the dsRNA has a 3′ overhang.
  • the antisense siRNA may be modified to include about 1 to about 9 ribonucleotides on the 5′ end to provide a length of about 22 to about 28 nucleotides.
  • the antisense strand has a length of 21 nucleotides, 1-7, preferably 2-5, or more preferably 4 ribonucleotides may be added on the 3′ end.
  • the added ribonucleotides may have any sequence.
  • the added ribonucleotides may be complementary to the target gene sequence, full complementarity between the target sequence and the antisense siRNA is not required. That is, the resultant antisense siRNA is sufficiently complementary with the target sequence.
  • a sense strand is then produced that has about 24 to about 30 nucleotides. The sense
  • 67 78645288V.1 strand is substantially complementary with the antisense strand to anneal to the antisense strand under biological conditions.
  • the antisense strand is synthesized to contain a modified 3′ end to direct Dicer processing.
  • the sense strand of the dsRNA has a 3′ overhang.
  • the antisense strand is synthesized to contain a modified 3′ end for Dicer binding and processing and the sense strand of the dsRNA has a 3′ overhang.
  • the nucleic acid of a provided lipid nanoparticle includes or consists of piRNA.
  • the piRNA molecule bind to proteins of A piRNA molecule can be about 10 to 50 nucleotides in length, about 25 to 39 nucleotides in length, or about 26 to 31 nucleotides in length. See, e.g., U.S. Patent Application Pub. No.2009/0062228.
  • piRNA molecules represent the largest class of small non-coding RNA molecules.
  • piRNAs can be substantially complementary to a target gene, and can selectively form RNA- protein complexes through interactions with the Piwi or Aubergine subclasses of Argonaute proteins. These piRNA complexes have been linked to both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells, particularly those in spermatogenesis. They are distinct from microRNA (miRNA) in size (typically 24-31 nt rather than 21-24 nt), lack of sequence conservation, and increased complexity. However, like other small RNAs, piRNAs are thought to be involved in gene silencing, specifically the silencing of transposons. The majority of piRNAs are antisense to transposon sequences, suggesting that transposons are the piRNA target.
  • miRNA microRNA
  • piRNAs In mammals it appears that the activity of piRNAs in transposon silencing is most important during the development of the embryo, and in both C. elegans and humans, piRNAs are necessary for spermatogenesis. piRNA has a role in RNA silencing via the formation of an RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the nucleic acid of a provided lipid nanoparticle includes or consists of an antisense oligonucleotide directed to a target gene or sequence of interest.
  • antisense oligonucleotide or “antisense” as used herein include oligonucleotides that are complementary to a targeted polynucleotide sequence.
  • Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a chosen sequence.
  • Antisense RNA oligonucleotides prevent the translation of complementary RNA strands by binding to the RNA.
  • Antisense DNA oligonucleotides can be used to target a specific, complementary (coding or non-coding) RNA. If binding occurs, this DNA/RNA hybrid can be degraded by the enzyme RNase H.
  • antisense oligonucleotides comprise from about 10 to about 60 nucleotides or from about 15 to about 30 nucleotides.
  • the term also encompasses antisense oligonucleotides that may not be exactly complementary to the desired target gene.
  • the lipid nanoparticles described herein can be utilized in instances where non-target specific-activities are found with antisense, or where an antisense sequence containing one or more mismatches with the target sequence is the most preferred for a particular use.
  • An antisense oligonucleotide can contain natural nucleotides, as well as non-natural or modified nucleotides (e.g., a modified nucleobase, modified internucleoside linkage, and/or modified sugar such as those described herein).
  • Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis, and, consequently, can be used to specifically inhibit protein synthesis by a targeted gene. The efficacy of antisense oligonucleotides for inhibiting protein synthesis is well established.
  • polygalacturonase and the muscarine type 2 acetylcholine receptor are inhibited by antisense oligonucleotides directed to their respective mRNA sequences (see, U.S. Pat. Nos.5,739,119 and 5,759,829).
  • antisense constructs have also been described that inhibit and can be used to treat a variety of abnormal cellular proliferations, e.g., cancer (see, U.S. Pat. Nos. 5,747,470; 5,591,317; and 5,783,683). The disclosures of these references are herein incorporated by reference in their entirety for all purposes.
  • antisense oligonucleotides are known in the art and can be readily adapted to produce an antisense oligonucleotide that targets any polynucleotide sequence. Selection of antisense oligonucleotide sequences specific for a given target sequence is based upon analysis of the chosen target sequence and determination of secondary structure, Tm, binding energy, and relative stability. Antisense oligonucleotides may be selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell.
  • Highly preferred target regions of the mRNA include those regions at or near the AUG translation initiation codon and those sequences that are substantially complementary to 5′ regions of the mRNA.
  • These secondary structure analyses and target site selection considerations can be performed, for example, using v.4 of the OLIGO primer analysis software (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).
  • the antisense oligonucleotide component of the lipid particles described herein can be used to inhibit the expression or replication of a gene of interest.
  • Genes of interest are set forth above in the context of siRNA molecules and include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., liver diseases and disorders), genes associated with tumorigenesis and cell transformation (e.g., cancer), angiogenic genes, immunomodulator genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.
  • the antisense oligonucleotide can hybridize to a SARS-CoV-2 gene (e.g., orf1ab, S gene, ORF3a, E gene, M gene, ORF6, ORF7a, ORF8, N gene, or ORF10) and inhibit the expression or replication of the gene.
  • the nucleic acid of a provided lipid nanoparticle includes or consists of a morpholino oligonucleotide.
  • Morpholino oligonucleotides are non-ionic and function by an RNase H-independent mechanism.
  • Each of the 4 genetic bases (Adenine, Cytosine, Guanine, and Thymine/Uracil) of the morpholino oligonucleotides is linked to a 6- membered morpholine ring.
  • Morpholino oligonucleotides are made by joining the 4 different subunit types by, e.g., non-ionic phosphorodiamidate inter-subunit linkages. Morpholino oligonucleotides have many advantages including: complete resistance to nucleases (Antisense),
  • Morpholino oligonucleotides are typically composed of two more morpholino monomers containing purine or pyrimidine base-pairing moieties effective to bind, by base- specific hydrogen bonding, to a base in a polynucleotide, which are linked together by phosphorus-containing linkages, one to three atoms long, joining the morpholino nitrogen of one monomer to the 5′ exocyclic carbon of an adjacent monomer.
  • the purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil or thymine.
  • the synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337.
  • the nucleic acid of a provided lipid nanoparticle includes or consists of DNA.
  • the DNA encapsulated by the lipid nanoparticle can serve as a template for the production of RNA molecules, e.g., any of the RNA molecules described in Sections.
  • the DNA is a template for the production of mRNA molecules, where the mRNA molecules can encode one or more polypeptides endogenously or exogenously expressed within a cell to which the DNA is delivered.
  • the DNA is a template for the production of interfering RNA molecules that can function to modulate (e.g., downregulate or silence) the expression of one or more polypeptides within a cell to which the DNA is delivered.
  • the DNA encapsulated by the lipid nanoparticle may have one or more of various structural and functional designs, including single stranded DNA, double stranded DNA, linear DNA, and circular DNA.
  • the DNA of a provided lipid nanoparticle has the form of a DNA construct having multiple (e.g., two or more) elements.
  • the DNA construct can, for example, include at least one element in addition to a DNA region serving as a template for the production of any of the RNA molecules described in Sections F.1 to F.8.
  • the one or more additional elements of the DNA construct enable the generation, function,
  • the one or more additional elements of the DNA construct include a promoter, a reporter, an enhancer, a transcription terminator, a polyadenylation sequence, a post-transcriptional regulatory element (PRE), or any combination of these features.
  • the DNA construct includes a promoter operably linked to a DNA region serving as a template for the production of an RNA molecule, e.g., any of the RNA molecules described in Sections F.1 to F.8. Numerous promoters may be used in the constructs described herein.
  • the promoter may be a eukaryotic or a prokaryotic promoter.
  • the DNA construct may include constitutive, inducible, tissue-preferred, or other promoters for expression in the organism or cell type of interest.
  • the promoter is a promoter capable of initiating transcription in hepatic stellate cells.
  • a promoter, or an active fragment thereof may be employed that will direct expression of a nucleic acid encoding a polypeptide in all transformed cells or tissues of a subject organism.
  • Such promoters are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation.
  • Exemplary constitutive promoters include PGK, EF1a core promoter (EF-S) SV40, CMV, UBC, EF1A, and CAGG.
  • the promoter or active fragment thereof is not always active and is referred to as an “inducible” promoter.
  • exemplary inducible promoters include chemically regulated promoters and physically regulated promoters.
  • Chemically regulated promoters include, for example, alcohol-regulated promoters (e.g., an alcohol dehydrogenase (alcA) gene promoter), tetracycline-regulated promoters (e.g., a tetracycline-responsive promoter, a tetracycline operator sequence (tetO), a tet-On promoter, or a tet-Off promoter), steroid regulated promoters (e.g., a rat glucocorticoid receptor, a promoter of an estrogen receptor, or a promoter of an ecdysone receptor), or metal-regulated promoters (e.g., a metalloprotein promoter).
  • alcohol-regulated promoters e.g., an
  • Physically regulated promoters include, for example temperature-regulated promoters (e.g., a heat shock promoter) and light-regulated promoters (e.g., a light-inducible promoter or a light-repressible promoter).
  • the DNA construct includes a reporter sequence encoding a reporter polypeptide that produces a detectable signal when the reporter polypeptide is expressed by the target cell.
  • the reporter provides an indication of the expression of a target gene or protein.
  • the reporter provides an indication of transformation of a target cell with the DNA construct.
  • the DNA construct reporter sequence includes a promoter that modulates expression of the reporter gene and is
  • the reporter promoter is a constitutive promoter. In other examples, the reporter promoter is an inducible promoter. In some examples, the reporter polypeptide is a fluorescent protein. In certain examples, the fluorescent protein is a green fluorescent protein (GFP), enhanced GFP (eGFP), TurboGFP, red fluorescent protein (RFP), tdTomato, mCherry, yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), or blue fluorescent protein (BFP).
  • GFP green fluorescent protein
  • eGFP enhanced GFP
  • TurboGFP red fluorescent protein
  • tdTomato mCherry
  • YFP yellow fluorescent protein
  • CFP cyan fluorescent protein
  • BFP blue fluorescent protein
  • the DNA of a provided lipid nanoparticle has the form of a DNA plasmid, a circular DNA molecule that is capable of independent replication and transcription.
  • Plasmid DNA can offer several advantages for lipid nanoparticle encapsulation and delivery, including stability, ease of production, and the ability to include multiple genetic elements in a single construct.
  • the plasmid may be selected, designed, or configured to allow introduction into the appropriate host cell, e.g., a hepatic stellate cell.
  • the plasmid may include one or more promoters, as well as other regulatory regions including, but not limited to, activator sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, ribosomal binding sites, and introns.
  • the plasmid may further include a marker gene that confers a selectable phenotype.
  • the marker may encode biocide resistance, particularly antibiotic resistance, such as those associated with neomycin phosphotransferase II (NEO), kanamycin, G418, bleomycin, and/or hygromycin phosphotransferase (HPT).
  • the plasmid may have a compact backbone with a length of no more than about 500 base pairs (bp), e.g., no more than about 450 bp, no more than about 400 bp, no more than about 350 bp, no more than about 300 bp, no more than about 250 bp, no more than about 200 bp, no more than about 150 bp, or no more than about 100 bp. Reducing the backbone length minimizes the overall size of the plasmid, which can improve the encapsulation efficiency and cellular uptake of the lipid nanoparticle.
  • bp base pairs
  • the plasmid lacks an antibiotic resistance marker.
  • the lack of antibiotic resistance marker can allow the plasmid to better address certain safety and regulatory concerns.
  • Traditional antibiotic resistance markers, such as genes for ampicillin or kanamycin resistance, may in some instances pose risks of horizontal gene transfer or
  • the plasmid may include alternative selection markers, such as auxotrophic markers, fluorescent reporters (e.g., GFP or mCherry), or toxin-antitoxin systems, to facilitate plasmid propagation and selection during production without relying on antibiotics.
  • the plasmid encapsulated by the lipid nanoparticle is a nanoplasmid, a specialized form of plasmid DNA with an optimized design for enhanced delivery and expression. Nanoplasmids are characterized by their minimal size, typically less than about 2.5 kilobases (kb) or less than about 500 bp, and the absence of unnecessary elements, such as origins of replication or antibiotic resistance markers.
  • Nanoplasmids may include optimized promoter and enhancer elements for high-level transcription of RNA editing system components in a wide range of cell types. Additionally, nanoplasmids may be engineered with sequences that enhance nuclear localization or minimize epigenetic silencing, ensuring robust and sustained RNA and/or protein production after delivery. 10. Target Genes of Downregulating Nucleic Acids [0203]
  • the nucleic acid component e.g., siRNA
  • the lipid nanoparticles described herein can be used to downregulate or silence the translation (i.e., expression) of a gene of interest.
  • Genes of interest include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., liver diseases and disorders), genes associated with tumorigenesis and cell transformation (e.g., cancer), angiogenic genes, immunomodulator genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.
  • the gene of interest is expressed in hepatic stellate cells.
  • Genes associated with viral infection and survival include those expressed by a virus in order to bind, enter, and replicate in a cell. Of particular interest are viral sequences associated with chronic viral diseases.
  • Viral sequences of particular interest include sequences of Filoviruses such as Ebola virus and Marburg virus (see, e.g., Geisbert et al., J. Infect. Dis., 193: 1650-1657 (2006)); Arenaviruses such as Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus (Buchmeier et al., Arenaviridae: the viruses and their
  • Influenza viruses such as Influenza A, B, and C viruses, (see, e.g., Steinhauer et al., Annu Rev Genet., 36:305-332 (2002); and Neumann et al., J Gen Viral., 83:2635-2662 (2002)); Hepatitis viruses (see, e.g., Hamasaki et al., FEBS Lett., 543:51 (2003); Yokota et al., EMBO Rep., 4:602 (2003); Schlomai et al., Hepatology, 37:764 (2003); Wilson et al., Proc.
  • Exemplary Filovirus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences encoding structural proteins (e.g., VP30, VP35, nucleoprotein (NP), polymerase protein (L-pol)) and membrane-associated proteins (e.g., VP40, glycoprotein (GP), VP24).
  • structural proteins e.g., VP30, VP35, nucleoprotein (NP), polymerase protein (L-pol)
  • membrane-associated proteins e.g., VP40, glycoprotein (GP), VP24.
  • Ebola virus VP24 sequences are set forth in, e.g., Genbank Accession Nos. U77385 and AY058897.
  • Ebola virus Lpol sequences are set forth in, e.g., Genbank Accession No. X67110.
  • Ebola virus VP40 sequences are set forth in, e.g., Genbank Accession No.
  • Ebola virus NP sequences are set forth in, e.g., Genbank Accession No. AY058895.
  • Ebola virus GP sequences are set forth in, e.g., Genbank Accession No. A Y058898; Sanchez et al., Virus Res., 29:215- 240 (1993); Will et al., J. Viral., 67:1203-1210 (1993); Volchkov et al., FEBS Lett., 305:181- 184 (1992); and U.S. Pat. No. 6,713,069.
  • Additional Ebola virus sequences are set forth in, e.g., Genbank Accession Nos. Ll 1365 and X61274.
  • Marburg virus GP sequences are set forth in, e.g., Genbank Accession Nos. AF005734; AF005733; and AF005732.
  • Marburg virus VP35 sequences are set forth in, e.g., Genbank Accession Nos. AF005731 and AF005730. Additional Marburg virus sequences are set forth in, e.g., Genbank Accession Nos. X64406; Z29337; AF005735; and Z12132.
  • Influenza virus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences encoding nucleoprotein (NP), matrix proteins (M 1 and M2), nonstructural proteins (NSl and NS2), RNA polymerase (PA, PBl, PB2), neuraminidase (NA), and haemagglutinin (HA).
  • NP nucleoprotein
  • M 1 and M2 matrix proteins
  • NSl and NS2 nonstructural proteins
  • NA neuraminidase
  • HA haemagglutinin
  • Influenza A NP sequences are set forth in, e.g., Genbank Accession Nos.
  • Influenza A PA sequences are set forth in, e.g., Genbank Accession Nos. AY818132; A Y790280; AY646171; AY818132; AY818133; AY646179; AY818134; AY551934; AY651613; AY651610; AY651620; AY651617; AY651600; AY651611; AY651606; AY651618; AY651608; AY651607; AY651605; AY651609; AY651615; AY651616; AY651640; AY651614; AY651612; AY651621; AY651619; AY770995; and AY724786.
  • Non-limiting examples of siRNA molecules targeting Influenza virus nucleic acid sequences include those described in U.S. Patent Application Publication No. 2007/0218122, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • Exemplary hepatitis virus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences involved in transcription and translation (e.g., En1, En2, X, P) and nucleic acid sequences encoding structural proteins (e.g., core proteins including C and C-related proteins, capsid and envelope proteins including S, M, and/or L proteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY, supra).
  • HCV nucleic acid sequences that can be silenced include, but are not limited to, the 5′ untranslated region (5′ UTR), the 3′ untranslated region (3′-UTR), the polyprotein translation initiation codon region, the internal ribosome entry site (IRES) sequence, and/or nucleic acid sequences encoding the core protein, the El protein, the E2 protein, the p7 protein, the NS2 protein, the NS3 protease/helicase, the NS4A protein, the NS4B protein, the NS5A protein, and/or the NS5B RNA-dependent RNA polymerase.
  • HCV genome sequences are set forth in, e.g., Genbank Accession Nos.
  • NC-004102 (HCV genotype la), AJ238799 (HCV genotype lb), NC-009823 (HCV genotype 2), NC-009824 (HCV genotype 3), NC-009825 (HCV genotype 4), NC-009826 (HCV genotype 5), and NC-009827 (HCV genotype 6).
  • HCV genotype la AJ238799
  • HCV genotype lb NC-009823
  • NC-009824 (HCV genotype 3)
  • NC-009825 (HCV genotype 4
  • NC-009826 (HCV genotype 5)
  • NC-009827 (HCV genotype 6).
  • nucleic acid sequences are set forth in, e.g., Genbank Accession No. NC-001489; Hepatitis B virus nucleic acid sequences are set forth in, e.g., Genbank Accession No. NC-003977; Hepatitis D virus nucleic acid sequence are set forth in, e.g., Genbank Accession No. NC- 001653; Hepatitis E virus nucleic acid sequences are set forth in, e.g., Genbank Accession No. NC-001434; and Hepatitis G virus nucleic acid sequences are set forth in, e.g., Genbank Accession No. NC-001710.
  • siRNA molecules targeting hepatitis virus nucleic acid sequences include those described in U.S. Patent Application Publication Nos. 2006/0281175, 2005/0058982, and 2007/0149470; U.S. Pat. No.7,348,314; and U.S. Provisional Application No.61/162,127, filed Mar.20, 2009, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • Genes associated with metabolic diseases and disorders include, for example, genes expressed in dyslipidemia (e.g., liver X receptors such as LXR ⁇ and LXR ⁇ (Genbank Accession No. NM- 007121), farnesoid X receptors (FXR) (Genbank Accession No. NM-005123), sterol- regulatory element binding protein (SREBP), site-1 protease (SIP), 3-hydroxy-3- methylglutaryl coenzyme A reductase (HMG coenzyme-A reductase), apolipoprotein B (ApoB) (Genbank Accession No.
  • dyslipidemia e.g., liver X receptors such as LXR ⁇ and LXR ⁇ (Genbank Accession No. NM- 007121), farnesoid X receptors (FXR) (Genbank Accession No. NM-005123), sterol- regulatory element binding protein (SREBP), site-1 protease (SIP),
  • apolipoprotein CIII Genbank Accession Nos. NM-000040 and NG- 008949 REGION: 5001.8164
  • apolipoprotein E Genbank Accession Nos. NM- 000041 and NG-007084 REGION: 5001.8612
  • diabetes e.g., glucose 6-phosphatase
  • genes associated with metabolic diseases and disorders include genes that are expressed in the liver itself as well as and genes expressed in other organs and tissues. Silencing of sequences that encode genes associated with metabolic diseases and disorders can conveniently be used in combination with the administration of conventional agents used to treat the disease or disorder.
  • Non-limiting examples of siRNA molecules targeting the ApoB gene include those described in U.S. Patent
  • siRNA molecules targeting the ApoC3 gene include those described in U.S. Provisional Application No.61/147,235, filed Jan.26, 2009, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • Examples of gene sequences associated with tumorigenesis and cell transformation include mitotic kinesins such as Eg5 (KSP, KIFl l; Genbank Accession No.
  • NM-004523 serine/threonine kinases such as polo-like kinase 1 (PLK-1) (Genbank Accession No. NM-005030; Barr et al., Nat. Rev. Mol. Cell. Biol., 5:429-440 (2004)); tyrosine kinases such as WEEl (Genbank Accession Nos. NM-003390 and NM- 001143976); inhibitors of apoptosis such as XIAP (Genbank Accession No. NM-001167); COP9 signalosome subunits such as CSNl, CSN2, CSN3, CSN4, CSN5 (JABl; Genbank Accession No.
  • RNA molecules targeting the Eg5 and XIAP genes include those described in U.S. patent application Ser. No. 11/807,872, filed May 29, 2007, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • Non-limiting examples of siRNA molecules targeting the PLK-1 gene include those described in U.S. Patent Application Publication Nos. 2005/0107316 and 2007/0265438; and U.S. Patent Application Ser. No.12/343,342, filed Dec. 23, 2008, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • Nonlimiting examples of siRNA molecules targeting the CSN5 gene include those described in U.S. Provisional Application No. 61/045,251, filed Apr. 15, 2008, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • translocation sequences such as MLL fusion genes, BCR-ABL (Wilda et al., Oncogene, 21:5716 (2002); Scherr et al., Blood, 101:1566 (2003)), TEL-AMLl, EWS- FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AMLl-ETO, and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003)); overexpressed sequences such as multidrug resistance genes (Nieth et al., FEBS Lett., 545:144 (2003); Wu et al, Cancer Res.
  • MLL fusion genes such as MLL fusion genes, BCR-ABL (Wilda et al., Oncogene, 21:5716 (2002); Scherr et al., Blood, 101:1566 (2003)), TEL-AMLl, EWS- FLI1, TLS-FUS, PAX3-
  • EGFR/ErbB1 Genbank Accession Nos. NM-005228, NM-201282, NM-201283, and NM-201284; see also, Nagy et al. Exp. Cell Res., 285:39-49 (2003), ErbB2/HER-2 (Genbank Accession Nos. NM-004448 and NM-001005862), ErbB3 (Genbank Accession Nos. NM-001982 and NM-001005915), and ErbB4 (Genbank Accession Nos.
  • siRNA molecules targeting the EGFR gene include those described in U.S. patent application Ser. No.11/807,872, filed May 29, 2007, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • Silencing of sequences that encode DNA repair enzymes find use in combination with the administration of chemotherapeutic agents (Collis et al., Cancer Res., 63: 1550 (2003)).
  • Genes encoding proteins associated with tumor migration are also target sequences of interest, for example, integrins, selectins, and metalloproteinases.
  • VEGF vascular endothelial growth factor
  • VEGFR vascular endothelial growth factor
  • siRNA sequences that target VEGFR are set forth in, e.g., GB 2396864; U.S. Patent Application Publication No. 2004/0142895; and CA 2456444, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • Anti-angiogenic genes are able to inhibit neovascularization. These genes are particularly useful for treating those cancers in which angiogenesis plays a role in the pathological development of the disease.
  • anti-angiogenic genes include, but are not limited to, endostatin (see, e.g., U.S. Pat. No.6,174,861), angiostatin (see, e.g., U U.S. Pat. No.5,639,725), and VEGFR2 (see, e.g., Decaussin et al., J. Pathol., 188: 369-377 (1999)), the disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • Immunomodulator genes are genes that modulate one or more immune responses.
  • immunomodulator genes include, without limitation, cytokines such as growth factors (e.g., TGF- ⁇ , TGF- ⁇ , EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4, IL-12 (Hill et al., J. Immunol., 171:691 (2003)), IL-15, IL-18, IL- 20, etc.), interferons (e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , etc.) and TNF.
  • Fas and Fas ligand genes are examples of cytokines such as growth factors (e.g., TGF- ⁇ , TGF- ⁇ , EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g.,
  • 79 78645288V.1 also immunomodulator target sequences of interest (Song et al., Nat. Med., 9:347 (2003)).
  • Genes encoding secondary signaling molecules in hematopoietic and lymphoid cells are also included in the present disclosure, for example, Tee family kinases such as Bruton’s tyrosine kinase (Btk) (Heinonen et al., FEBS Lett., 527:274 (2002)).
  • Cell receptor ligands include ligands that are able to bind to cell surface receptors (e.g., insulin receptor, EPO receptor, G-protein coupled receptors, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.), to modulate (e.g., inhibit, activate, etc.) the physiological pathway that the receptor is involved in (e.g., glucose level modulation, blood cell development, mitogenesis, etc.).
  • cell receptor ligands include, but are not limited to, cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G-protein coupled receptor ligands, etc.
  • Templates coding for an expansion of trinucleotide repeats find use in silencing pathogenic sequences in neurodegenerative disorders caused by the expansion of trinucleotide repeats, such as spinobulbular muscular atrophy and Huntington’s Disease (Caplen et al., Hum. Mol. Genet., 11:175 (2002)).
  • Certain other target genes which may be targeted by a nucleic acid (e.g., by siRNA) to downregulate or silence the expression of the gene, include but are not limited to, Actin, Alpha 2, Smooth Muscle, ACTA2, Alcohol dehydrogenase IA (ADRIA), Alcohol dehydrogenase 4 (ADH4), Alcohol dehydrogenase 6 (ADH6), Afamin (AFM), Angiotensinogen (AGT), Serine-pyruvate aminotransferase (AGXT), Alpha-2-HS- glycoprotein (AHSG), Aldoketo reductase family I member C4 (AKRIC4), Serum albumin (ALB), alpha-1- microglobulin/bikunin precursor (AMBP), Angiopoietin-related protein 3 (ANGPTL3), Serum amyloid P-component (APCS), Apolipoprotein A-II (APOA2), Apolipoprotein B-100 (APOB), Apoli
  • carboxypeptidase B2 (CPB2), Connective tissue growth factor (CTGF), C-X-C motif chemokine 2 (CXCL2), Cytochrome P4501A2 (CYP1A2), Cytochrome P4502A6 (CYP2A6), Cytochrome P450 2C8 (CYP2C8), Cytochrome P450 2C9 (CYP2C9), Cytochrome P450 Family 2 Subfamily D Member 6 (CYP2D6), Cytochrome P4502El (CYP2El), Phylloquinone omega-hydroxylase CYP4F2 (CYP4F2), 7-alpha-hydroxycholest-4-en-3-one 12-alpha- hydroxylase (CYP8B 1), Dipeptidyl peptidase 4 (DPP4), coagulation factor 12 (Fl2), coagulation factor II (thrombin) (F2), coagulation factor IX (F9), fibrinogen alpha chain (F
  • the siRNA is configured to reduce expression of a gene in a hepatic stellate cell.
  • the siRNA can reduce expression of a gene encoding collagen type I alpha 1 chain (COL1A1), COL1A2, COL2A1, COL3A1, COL4A1, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL7A1, COL8A2, COL9A1, COL9A2, COL9A3, COL10A1, COL11A1, COL11A2, COL17A1, COL18A1, tissue inhibitor of metalloproteinases 1 (TIMP1), matrix metalloproteinase 2 (MMP2), MMP1, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP22, MMP23, MMP24, MMP25, MMP26, MMP28, al
  • nucleic acids can be used in target validation studies directed at testing whether a gene of interest has the potential to be a therapeutic target.
  • Certain nucleic acids e.g., siRNA
  • target identification studies aimed at discovering genes as potential therapeutic targets.
  • the disclosure provides a pharmaceutical composition that include one or more of any of the lipid nanoparticles described herein in Section III.
  • the pharmaceutical composition further includes a therapeutically effective amount of a pharmaceutically acceptable excipient.
  • the pharmaceutical composition can include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier e.g., physiological saline or phosphate buffer
  • physiological saline or phosphate buffer can be selected in accordance with the route of administration and standard pharmaceutical practice.
  • normal buffered saline e.g., 135-150 mM NaCl
  • 82 78645288V.1 as the pharmaceutically acceptable carrier.
  • suitable carriers include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. Additional suitable carriers are described in, e.g., Remington: The Science and Practice of Pharmacy, 23rd Edition (2020).
  • the pharmaceutically acceptable carrier is generally added following particle formation. Thus, after the particle is formed, the particle can be diluted into pharmaceutically acceptable carriers such as normal buffered saline.
  • excipients for use with the provided pharmaceutical compositions include, but are not limited to, lactose, dextrose, sucrose, maltose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc.
  • Carbopols e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc.
  • the provided pharmaceutical compositions can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preserving agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents such as inorganic and organic acids and bases; sweetening agents; coloring agents; and flavoring agents.
  • lubricating agents such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifying agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the parabens)
  • pH adjusting agents such as inorganic and organic acids and bases
  • sweetening agents coloring agents
  • flavoring agents can also comprise biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.
  • compositions that are liquid compositions, whether they are solutions, suspensions or other like form can also include one or more of the following: sterile diluents such as water for injection, saline solution (preferably physiological saline), Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as amino acids, acetates, citrates or phosphates; detergents, such as nonionic surfactants, polyols; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • sterile diluents such as water for injection, saline solution
  • the liquid compositions can include aqueous or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles.
  • Aqueous solutions in saline are also conventionally used for injection. Ethanol, glycerol,
  • compositions for administration are preferably sterile.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid and thimerosal.
  • compositions for administration can be enclosed in ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic, or other material.
  • the provided pharmaceutical compositions can be in the form of tablets, lozenges, capsules, emulsions, suspensions, solutions, syrups, sprays, powders, and sustained-release formulations.
  • Suitable excipients for oral administration include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.
  • the provided pharmaceutical compositions can be in the form of emulsions, lotions, gels, creams, jellies, solutions, suspensions, ointments, and transdermal patches.
  • the composition can be delivered as a dry powder or in liquid form via a nebulizer.
  • the compositions can be in the form of sterile injectable solutions and sterile packaged powders. Injectable solutions can be formulated at a pH of about 4.5 to about 8.5, and preferably at a pH between about 6.0 and about 7.5.
  • the concentration of lipid nanoparticles in the provided pharmaceutical formulations can vary widely, e.g., from less than about 0.05%, usually at or at least about 2 to about 5%, to as much as about 10 to about 90% by weight, and can be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. This may be particularly desirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, lipid nanoparticles composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration. [0228]
  • the provided pharmaceutical compositions can be sterilized by conventional, well- known sterilization techniques. Aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile
  • compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride.
  • lipid nanoparticle suspension can include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free- radical quenchers, such as alphatocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable. V.
  • Materials and reagents to carry out these various methods can be provided in kits to facilitate execution of the methods.
  • the provided kits can contain chemical reagents as well as other components.
  • the kits containing the provided lipid nanoparticles and/or pharmaceutical compositions can include, without limitation, instructions to the kit user.
  • Kits can also be packaged for convenient storage and safe shipping, for example, as ampules or other vials packaged in a box having a lid.
  • the provided kits can include one or more containers, each of which can be compartmentalized for holding the various elements of the lipid nanoparticles (e.g., the nucleic acids and the individual lipid components of the nanoparticles).
  • the kit further includes an endosomal membrane destabilizer (e.g., calcium ions).
  • the kit typically contains the lipid nanoparticle compositions of the present disclosure, preferably in dehydrated form, with instructions for their rehydration and administration. VI.
  • the disclosure provides several methods for making and/or using the lipid nanoparticles described in Section III, the pharmaceutical compositions described in Section IV, and/or the kits described in Section V.
  • the methods disclosed herein benefit from the improved properties of these provided materials, e.g., properties advantageous for delivering a nucleic acid into a cell (e.g., a hepatic stellate cell).
  • lipid nanoparticles include those described in Section III.
  • the lipid nanoparticles can be prepared via, for example, a continuous mixing method and/or a direct dilution process.
  • the methods include selecting, designing, and/or preparing one or more components of the lipid nanoparticle being prepared.
  • the methods can include selecting, designing, and/or preparing a phospholipid, an ionizable lipid, a cholesterol or derivative thereof, a conjugated lipid, and/or a nucleic acid.
  • the lipid nanoparticles described herein are produced via a continuous mixing method, e.g., a process that includes providing an aqueous solution comprising a nucleic acid (e.g., any of the nucleic acids described in Section III.F) in a first reservoir, providing an organic lipid solution in a second reservoir, and mixing the aqueous solution with the organic lipid solution such that the organic lipid solution mixes with the aqueous solution so as to substantially instantaneously produce a lipid particle encapsulating the nucleic acid.
  • a continuous mixing method e.g., a process that includes providing an aqueous solution comprising a nucleic acid (e.g., any of the nucleic acids described in Section III.F) in a first reservoir, providing an organic lipid
  • the organic lipid solution is an alcoholic solution that includes an alcoholic organic solvent and the lipids that will form the lipid nanoparticles.
  • These lipids can include one or more permanently charged cationic lipids or pharmaceutically acceptable salts thereof, one or more conjugated lipids, one or more ionizable lipids or pharmaceutically acceptable salts thereof, one or more sterols or derivatives thereof, and one or more phospholipids or pharmaceutically acceptable salts thereof.
  • the permanently charged cationic lipid can be any of those described in Section III.A
  • the phospholipid can be any of those described in Section III.C
  • the conjugated lipid can be any of those described in Section III.E
  • the ionizable lipid can be any of those described in Section III.B
  • the sterol can be cholesterol or any of its derivatives described in Section III.D.
  • the alcoholic organic solvent of the alcoholic solution includes or consists of ethanol.
  • the alcoholic solution is the product of combining the alcoholic organic solvent and a phospholipid solution, where the phospholipid solution includes or consists of a nonalcoholic
  • the nonalcoholic organic solvent includes or consists of tetrahydrofuran.
  • the organic lipid solution undergoes a continuous stepwise dilution in the presence of the buffer solution (i.e., aqueous solution) to produce a lipid particle.
  • the buffer solution i.e., aqueous solution
  • the lipid nanoparticles formed using the continuous mixing method typically have a size of from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 60 nm to about 100 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 60 to about 90 nm, or from about 70 nm to about 90 nm.
  • the particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.
  • the lipid nanoparticles described herein are produced via a direct dilution process that includes forming a lipid particle solution and immediately and directly introducing the lipid particle solution into a collection vessel containing a controlled amount of dilution buffer.
  • the collection vessel includes one or more elements configured to stir the contents of the collection vessel to facilitate dilution.
  • the amount of dilution buffer present in the collection vessel is substantially equal to the volume of lipid particle solution introduced thereto.
  • a lipid particle solution in 45% ethanol when introduced into the collection vessel containing an equal volume of dilution buffer will advantageously yield smaller particles.
  • the lipid nanoparticles described herein are produced via a direct dilution process in which a third reservoir containing dilution buffer is fluidly coupled to a second mixing region.
  • the lipid particle solution formed in a first mixing region is immediately and directly mixed with dilution buffer in the second mixing region.
  • the second mixing region includes a T-connector arranged so that the lipid particle solution and the dilution buffer flows meet as opposing 180 o flows;
  • a pump mechanism delivers a controllable flow of buffer to the second mixing region.
  • the flow rate of dilution buffer provided to the second mixing region is controlled to be substantially equal to the flow rate of lipid particle solution introduced thereto from the first mixing region.
  • This embodiment advantageously allows for more control of the flow of dilution buffer mixing with the lipid particle solution in the second mixing region, and therefore also the concentration of lipid particle solution in buffer throughout the second mixing process.
  • Such control of the dilution buffer flow rate advantageously allows for small particle size formation at reduced concentrations.
  • the lipid particles formed using the direct dilution process typically have a size of from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 60 nm to about 100 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 60 to about 90 nm, or from about 70 nm to about 90 nm.
  • the particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.
  • the lipid particles described herein can be sized by any of the methods available to one of skill in the art.
  • the sizing may be conducted in order to achieve a desired size range and relatively narrow distribution of particle sizes.
  • Several techniques are available for sizing the particles to a desired size.
  • One sizing method, used for liposomes and equally applicable to the present particles, is described in U.S. Patent No. 4,737,323. Sonicating a particle suspension either by bath or probe sonication produces a progressive size reduction down to particles of less than about 50 nm in size. Homogenization is another method which relies on shearing energy to fragment larger particles into smaller ones.
  • particles are recirculated through a standard emulsion homogenizer until selected particle sizes, typically between about 60 and about 80 nm, are observed.
  • the particle size distribution can be monitored by conventional laser-beam particle size discrimination, or QELS.
  • Extrusion of the particles through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing particle sizes to a relatively well-defined size distribution.
  • the suspension is cycled through the
  • the nucleic acid to lipid ratios (mass/mass ratios) in a formed lipid particle ranges from about 0.01 to about 0.2, from about 0.02 to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about 0.08.
  • the ratio of the starting materials can also fall within this range.
  • the lipid to nucleic acid ratio (mass/mass ratios) in a formed lipid particle ranges from about 5 to about 100, from about 8 to about 50, from about 9 to about 30, or from about 10 to about 20. In other embodiments, the lipid particle preparation includes about 12 mg total lipid per 1 mg of nucleic acid.
  • the lipid to nucleic acid ratios (mass/mass ratios) in a formed lipid particle ranges from about 1 (1:1) to about 100 (100:1), from about 5 (5:1) to about 100 (100:1), from about 1 (1:1) to about 50 (50:1), from about 2 (2:1) to about 50 (50:1), from about 3 (3:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 5 (5:1) to about 50 (50:1), from about 1 (1:1) to about 25 (25:1), from about 2 (2:1) to about 25 (25:1), from about 3 (3:1) to about 25 (25:1), from about 4 (4:1) to about 25 (25:1), from about 5 (5:1) to about 25 (25:1), from about 5 (5:1) to about 20 (20:1), from about 5 (5:1) to about 15 (15:1), from about 5 (5:1) to about 10 (10:1), about 5 (5:1), 6 (6:1),
  • Some methods provided by the disclosure are useful for reducing expression of a gene in a cell, particularly a hepatic stellate cell, by introducing a nucleic acid into the cell, where the nucleic acid is configured to reduce the gene expression. These methods generally include contacting the cell with one or more lipid nanoparticles described in Section III.
  • the nucleic acid introduced to the cell using the lipid nanoparticle can be, for example, any of those described in Section III.F.
  • the lipid nanoparticles described herein are particularly useful for the introduction of nucleic acids such as siRNA into cells.
  • the methods are carried out in vitro or in vivo by first forming the particles and then contacting the particles with the cells for a period of time sufficient for delivery of the nucleic acid to the cells to occur.
  • the permanently charged cationic lipids and lipid nanoparticles provided by the current disclosure are particularly useful for preferentially delivering nucleic acids, such as RNA (e.g., interfering RNA), to hepatic stellate cells of a subject.
  • nucleic acids such as RNA (e.g., interfering RNA)
  • the specific permanently charged cationic lipids and lipid nanoparticle configurations described in Section III can advantageously result in materials exhibiting improved targeting of the hepatic stellate cells relative to other cells and organs of a subject to whom the materials are administered.
  • the provided permanently charged cationic lipids and lipid nanoparticle formulations can be beneficially applied to preferentially reducing expression of a target gene in hepatic stellate cells to a greater extent than expression of the target gene is reduced in other cells and organs.
  • the provided permanently charged cationic lipids and lipid nanoparticles can be used to deliver a nucleic acid reducing expression of a gene to hepatic stellate cells of a subject, such that the expression of the gene in the hepatic stellate cells is at least about 5-fold less than, e.g., at least about 7-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 70-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 500-fold, at least about 700-fold, or at least about 1000-fold less than an expression of the gene in hepatocytes of the subject under otherwise identical or essentially identical conditions.
  • the lipid nanoparticles described herein can preferentially be adsorbed to hepatic stellate cells with which they are mixed or contacted. Once adsorbed, the particles can either be endocytosed by a portion of the cells, exchange lipids with cell membranes, or fuse with the cells. Transfer or incorporation of the nucleic acid portion of the particle can take place via any one of these pathways. In particular, when fusion takes place, the particle membrane is integrated into the cell membrane and the contents of the particle combine with the intracellular fluid. [0248] Contact between the cells and the lipid nanoparticles, when carried out in vitro, generally takes place in a biologically compatible medium.
  • the amount of the composition used can vary widely depending on the particular application, but is generally between about 1 ⁇ mol and about 10 mmol.
  • Treatment of the cells with the lipid particles is generally carried out at physiological temperatures (about 37 °C) for periods of time ranging from about 1 hour to about 48 hours. In some examples, the contacting of the cells with the lipid nanoparticles is for a duration lasting from about 2 to about 4 hours.
  • a lipid nanoparticle suspension is added to 60-80% confluent plated cells having a cell density of from about 10 3 to about 10 5 cells/mL, e.g., about 2 ⁇ 10 4 cells/mL.
  • the concentration of the suspension added to the cells can be from about 0.01 to 0.2 ⁇ g/mL, e.g., about 0.1 ⁇ g/mL.
  • ERP Endosomal Release Parameter
  • an ERP assay is to distinguish the effect of various cationic lipids and helper lipid components based on their relative effect on binding/uptake or fusion with/destabilization of the endosomal membrane. This assay allows one to determine quantitatively how each component of the lipid particle affects delivery efficiency, thereby optimizing the lipid particle.
  • an ERP assay measures expression of a reporter protein (e.g., luciferase, ⁇ -galactosidase, green fluorescent protein (GFP), etc.), and in some instances, a lipid particle formulation optimized for an expression plasmid will also be appropriate for encapsulating other types of nucleic acid such as mRNA.
  • a reporter protein e.g., luciferase, ⁇ -galactosidase, green fluorescent protein (GFP), etc.
  • an ERP assay can be adapted to measure downregulation of transcription or translation of a target sequence in the presence or absence of an interfering RNA (e.g., siRNA).
  • an ERP assay can be adapted to measure the expression of a target protein in the presence or absence of an mRNA.
  • Suitable cells include, e.g., hepatic stellate cells, hematopoietic precursor (stem) cells, fibroblasts, keratinocytes, hepatocytes, immune cells, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, quiescent lymphocytes, terminally differentiated cells, slow or noncycling primary cells, parenchymal cells, lymphoid cells, epithelial cells, bone cells, and the like.
  • stem hematopoietic precursor
  • fibroblasts keratinocytes, hepatocytes, immune cells, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, quiescent lymphocytes, terminally differentiated cells, slow or noncycling primary cells, parenchymal cells, lymphoid cells, epithelial cells, bone cells, and the like.
  • lipid particles comprising nucleic acid are delivered to immune cells such as e.g., antigen-presenting cells (e.g., dendritic cells, macrophages, B cells) and T cells (e.g., helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells).
  • lipid particles comprising nucleic acid e.g., siRNA
  • cancer cells such as, e.g., lung cancer cells, colon cancer cells, rectal cancer cells, anal cancer cells, bile duct cancer cells, small intestine cancer
  • stomach (gastric) cancer cells esophageal cancer cells, gallbladder cancer cells, liver cancer cells, pancreatic cancer cells, appendix cancer cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, prostate cancer cells, renal cancer cells, cancer cells of the central nervous system, glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteogenic sarcoma tumor cells, and blood cancer cells.
  • nucleic acids e.g., mRNA and/or siRNA
  • the delivery of nucleic acids can be to any cell grown in culture, whether of plant or animal origin, vertebrate or invertebrate, and of any tissue or type.
  • the cells are animal cells, e.g., mammalian cells such as human cells.
  • in vivo delivery of the provided lipid particles such as lipid nanoparticles encapsulating one or more nucleic acid molecules (e.g., interfering RNA (e.g., siRNA) or mRNA) is particularly suited for targeting hepatic stellate cells.
  • tissue culture of cells may be required, it is well-known in the art.
  • the methods and compositions disclosed herein can be employed with cells of a wide variety of vertebrates, including mammals, such as, e.g., canines, felines, equines, bovines, ovines, caprines, rodents (e.g., mice, rats, and guinea pigs), lagomorphs, swine, and primates (e.g. monkeys, chimpanzees, and humans).
  • These methods generally include administering one or more lipid nanoparticles or one or more pharmaceutical compositions to the subject, wherein the lipid nanoparticles include those described in Section III, and the pharmaceutical compositions include those described in Section IV.
  • the nucleic acid delivered to the subject can be, for example, any of those described in Section III.F.
  • the lipid nanoparticles described herein are administered to a subject by systemic delivery, e.g., to a distal target cell via body systems such as the circulation.
  • the present disclosure provides fully encapsulated lipid particles that protect the nucleic acid from nuclease degradation in serum, are nonimmunogenic, are small in size, and are suitable for repeat dosing.
  • the lipid nanoparticles provided by the current disclosure are particularly useful for preferentially delivering nucleic acids, such as RNA, to hepatic stellate cells of a subject.
  • nucleic acids such as RNA
  • the specific permanently charged cationic lipids and lipid nanoparticle configurations described in Section IV advantageously result in materials exhibiting improved targeting of hepatic stellate cells relative to other cell types and organs of a subject to whom the materials are administered.
  • the provided lipid nanoparticles can be used to deliver a nucleic acid reducing expression of a protein by hepatic stellate cells of a subject, such that the activity or secretion of the protein in the hepatic stellate cells is more than about 20-fold reduced, more than about 30-fold, more than about 40-fold, more than about 60-fold, more than about 100-fold, more than about 200-fold, more than about 300-fold, more than about 500-fold, more than about 700-fold, or more than about 1000-fold reduced relative an activity or expression of the protein in the absence of the nucleic acid delivered by the provided lipid nanoparticles.
  • administration can be in any manner known in the art, e.g., by injection, oral administration, inhalation (e.g., intranasal or intratracheal), transdermal application, or rectal administration. Administration can be accomplished via single or divided doses.
  • the pharmaceutical compositions can be administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection (see, e.g., U.S. Patent No.5,286,634).
  • Intracellular nucleic acid delivery has also been discussed in Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino et al., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. Drug Carrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993). Still other methods of administering lipid-based therapeutics are described in, for example, U.S. Patent Nos.3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578.
  • the lipid nanoparticles can be administered by direct injection at the site of disease or by injection at a site distal from the site of disease (see, e.g., Culver, HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp. 70- 71(1994)).
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile
  • the compositions are administered intravenously (e.g., by intravenous infusion), intramuscularly, pulmonarily, orally, topically, intranasally, intracerebrally, intraperitoneally, intravesically, or intrathecally.
  • intravenously e.g., by intravenous infusion
  • intramuscularly pulmonarily, orally
  • topically intranasally, intracerebrally, intraperitoneally, intravesically, or intrathecally.
  • the lipid particle formulations are formulated with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier may be employed in the compositions and methods described herein. Suitable formulations for use are found, for example, in Adejare, A. (Ed.). (2020).
  • aqueous carriers may be used, for example, water, buffered water, 0.4% saline, 0.3% glycine, and the like, and may include glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc.
  • glycoproteins for enhanced stability such as albumin, lipoprotein, globulin, etc.
  • normal buffered saline (135-150 mM NaCl) is used as the pharmaceutically acceptable carrier, but other suitable carriers will suffice.
  • These compositions can be sterilized by conventional liposomal sterilization techniques, such as filtration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • hosts include mammalian species, such as primates (e.g., humans and chimpanzees as well as other nonhuman primates), canines, felines, equines, bovines, ovines, caprines, rodents (e.g., rats and mice), lagomorphs, and swine.
  • the amount of particles administered will depend upon the ratio of nucleic acid (e.g., mRNA) to lipid, the particular nucleic acid used, the disease or disorder being treated, the age, weight, and condition of the subject, and the judgment of the clinician, but will generally be
  • D. Preventing or Treating a Disease or Disorder Some methods provided by the disclosure are useful for preventing or treating a disease or a disorder of a subject. These methods generally include administering one or more lipid nanoparticles or one or more pharmaceutical compositions to the subject, where the lipid nanoparticles include those described in Section IV and the pharmaceutical compositions include those described in Section V. In some embodiments, the treating of the disease in the subject includes decreasing or eliminating one or more signs or symptoms of the disease.
  • the prevented or treated disease is a liver disease or disorder.
  • the prevented or treated liver disease or disorder can be, for example, a disease or disorder associated with hepatic stellate cells.
  • liver diseases or disorders that are associated with hepatic stellate cells and that can be prevented or treated with the provided lipid nanoparticles and/or pharmaceutical compositions include liver fibrosis, liver cirrhosis, hepatocellular carcinoma (HCC), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), alcoholic liver disease (ALD), immune-induced hepatitis (e.g., Concanavalin A (ConA)-induced hepatitis and lipopolysaccharide/D-galactosamine (LPS/GalN)-induced hepatitis), ischemia and reperfusion (I/R) liver injury, and schistosome and/or chronic viral
  • treatment with the provided lipid nanoparticles and/or pharmaceutical compositions results in stable disease, partial remission or complete remission in the subject (e.g., the methods described herein comprise administering to the subject a dose of the provided lipid nanoparticles and/or pharmaceutical compositions that kills or otherwise slows the growth or progression of cancer cells and leads to stable disease or to partial or complete remission of the cancer in the subject).
  • treatment with the provided lipid nanoparticles and/or pharmaceutical compositions results in a reduction in metastases of the cancer in the subject (e.g., the methods described herein comprise administering to the subject a dose of the provided lipid nanoparticles and/or pharmaceutical compositions that reduces metastases of the cancer in the subject).
  • treatment with the provided lipid nanoparticles and/or pharmaceutical compositions results in a reduction in volume, size, or growth of a tumor in the subject (e.g., the methods described herein comprise administering to the subject a dose of the provided lipid nanoparticles and/or
  • treatment with the provided lipid nanoparticles and/or pharmaceutical compositions results in an increased responsiveness of the cancer to a subsequently administered anti-cancer agent (e.g., the methods described herein comprise administering to the subject a dose of the provided lipid nanoparticles and/or pharmaceutical compositions that increases responsiveness of the cancer to a subsequently administered anti- cancer agent).
  • the provided method further includes obtaining a test sample from the subject.
  • the test sample can include, for example, a blood sample, a tissue sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, or a combination thereof.
  • the provided method further includes determining the level of one or more biomarkers in the obtained test sample. Determining the presence or level of biomarkers(s) can be used to, as non-limiting examples, determine response to treatment or to select an appropriate composition for the prevention or treatment of the disease.
  • the measured biomarker can be an indicator of an expression level of the protein or fragment by a target cell or target organism, e.g., the cell or organism to which the lipid nanoparticle or pharmaceutical composition is administered.
  • the biomarker can be the protein or fragment itself, such that a measured level of the protein or fragment indicates the level of its expression.
  • the measured biomarker can include or consist of one or more components of the immune system of the target cell or target organism.
  • the biomarker can include or consist of one or more species of T cells, e.g., CD8 + T cells.
  • the determination of a level of a biomarker in a test sample can provide information related to the efficiency and/or effectiveness of the administration of the lipid nanoparticle in preventing or treating a disease or disorder.
  • the provided method further includes comparing the determined level of the one of more biomarkers in the obtained test sample to the level of the one or more biomarkers in a reference sample.
  • the reference sample can be obtained, for example, from the subject, with the reference sample being obtained prior to the obtaining of the test sample, e.g., prior to the administering to the subject of the therapeutically effective amount of the provided materials. In this way, the reference sample can provide information
  • the reference sample can be obtained, for example, from a different subject, e.g., a subject in which the treatment is not provided according to the provided methods. In this way, the reference sample can provide information about baseline levels of the biomarkers without treatment, and the test sample can provide information about levels of the biomarkers with treatment.
  • the reference sample can also be obtained, for example, from a population of subjects, e.g., subjects in which the treatment is not provided according to the provided method.
  • the reference sample can provide population-averaged information about baseline levels of the biomarkers without treatment, and the test sample can provide information about levels of the biomarkers with treatment.
  • the reference sample can also be obtained from an individual or a population of individuals after treatment is provided according to the provided methods, and can serve as, for example, a positive control sample.
  • the reference sample is obtained from normal tissue.
  • the reference sample is obtained from abnormal tissue.
  • an increase or decrease relative to a normal control or reference sample can be indicative of the presence of a disease, or response to treatment for a disease.
  • an increased level of a biomarker in a test sample, and hence the presence of a disease, e.g., an infectious disease or cancer, increased risk of the disease, or response to treatment is determined when the biomarker levels are at least, 1.1-fold, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7- fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5- fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold higher in comparison to a control.
  • a disease e.g., an infectious disease or
  • a decreased level of a biomarker in the test sample, and hence the presence of the disease, increased risk of the disease, or response to treatment is determined when the biomarker levels are at least 1.1-fold, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at
  • biomarker levels can be detected using any method known in the art, including the use of antibodies specific for the biomarkers. Exemplary methods include, without limitation, polymerase chain reaction (PCR), Western Blot, dot blot, ELISA, radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, FACS analysis, electrochemiluminescence, and multiplex bead assays, e.g., using Luminex or fluorescent microbeads.
  • PCR polymerase chain reaction
  • RIA radioimmunoassay
  • immunoprecipitation immunofluorescence
  • FACS analysis electrochemiluminescence
  • electrochemiluminescence and multiplex bead assays, e.g., using Luminex or fluorescent microbeads.
  • nucleic acid sequencing is employed.
  • the presence of decreased or increased levels of one or more biomarkers is indicated by a detectable signal, e.g., a blot, fluorescence, chemiluminescence, color, or radioactivity in an immunoassay or PCR reaction, e.g., quantitative PCR.
  • a detectable signal e.g., a blot, fluorescence, chemiluminescence, color, or radioactivity in an immunoassay or PCR reaction, e.g., quantitative PCR.
  • This detectable signal can be compared to the signal from a reference sample or to a threshold value.
  • the results of the biomarker level determinations are recorded in a tangible medium.
  • the results of diagnostic assays e.g., the observation of the presence or decreased or increased presence of one or more biomarkers, and the diagnosis of whether or not there is an increased risk or the presence of a disease, e.g., an infectious disease or cancer, or whether or not a subject is responding to treatment can be recorded, for example, on paper or on electronic media, e.g., audio tape, a computer disk, a CD-ROM, or a flash drive.
  • the provided method further includes the step of providing to the subject a diagnosis and/or the results of treatment. VII. EXEMPLARY EMBODIMENTS [0275] The following embodiments are contemplated. All combinations of features and embodiments are contemplated.
  • Embodiment 1 A method for reducing expression of a gene in a hepatic stellate cell, the method comprising: contacting the hepatic stellate cell with a lipid nanoparticle comprising: a nucleic acid configured to reduce expression of the gene in the hepatic stellate cell; an ionizable lipid or a pharmaceutically acceptable salt thereof; a phospholipid or a pharmaceutically acceptable salt thereof; cholesterol or a derivative thereof; a conjugated lipid; and a permanently charged cationic lipid or a pharmaceutically acceptable salt thereof; thereby introducing the nucleic acid into the hepatic stellate cell and reducing expression of the gene in the hepatic stellate cell.
  • a lipid nanoparticle comprising: a nucleic acid configured to reduce expression of the gene in the hepatic stellate cell; an ionizable lipid or a pharmaceutically acceptable salt thereof; a phospholipid or a pharmaceutically acceptable salt thereof; cholesterol or
  • Embodiment 2 An embodiment of embodiment 1, wherein the gene encodes collagen type I alpha 1 chain (COL1A1), COL1A2, COL2A1, COL3A1, COL4A1, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL7A1, COL8A2, COL9A1, COL9A2, COL9A3, COL10A1, COL11A1, COL11A2, COL17A1, COL18A1, tissue inhibitor of metalloproteinases 1 (TIMP1), matrix metalloproteinase 2 (MMP2), MMP1, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP22, MMP23, MMP
  • Embodiment 3 An embodiment of embodiment 1 or 2, wherein the nucleic acid comprises RNA.
  • Embodiment 4 An embodiment of embodiment 3, wherein the RNA comprises siRNA, miRNA, shRNA, asRNA, DsiRNA, piRNA, or a combination thereof.
  • Embodiment 5 An embodiment of embodiment 4, wherein the RNA comprises siRNA.
  • Embodiment 6 An embodiment of embodiment 1 or 2, wherein the nucleic acid comprises an antisense oligonucleotide, a morpholino oligonucleotide, or a combination thereof.
  • Embodiment 7 An embodiment of any one of embodiments 1-6, wherein the permanently charged cationic lipid comprises: one or more quaternary ammonium nitrogens; a heteroatom branch point other than the one or more quaternary ammonium nitrogens, the heteroatom branch point directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members; and three or more terminal hydrocarbon chains not directly bonded to the one or more quaternary ammonium nitrogens, wherein the terminal hydrocarbon chains are each independently alkyl or alkenyl.
  • Embodiment 8 An embodiment of embodiment 7, wherein the heteroatom branch point is a silicon.
  • Embodiment 9 An embodiment of embodiment 7, wherein the heteroatom branch point is a nitrogen.
  • Embodiment 10 An embodiment of any one of embodiments 7-9, wherein the heteroatom branch point is directly bonded to at least one optionally oxo-substituted and optionally halogen-substituted heteroalkenyl group.
  • Embodiment 11 An embodiment of any one of embodiments 7-10, wherein the heteroatom branch point is directly bonded to at least one optionally oxo-substituted and optionally halogen-substituted alkenoxy group.
  • Embodiment 12 An embodiment of embodiment 11, wherein the heteroatom branch point is directly bonded to at least two optionally oxo-substituted and optionally halogen- substituted alkenoxy groups.
  • Embodiment 13 An embodiment of any one of embodiments 7-12, wherein the heteroatom branch point is directly bonded to at least one optionally oxo-substituted and optionally halogen-substituted alkyl group.
  • Embodiment 14 An embodiment of embodiment 13, wherein the heteroatom branch point is directly bonded to at least two optionally oxo-substituted and optionally halogen- substituted alkyl groups.
  • Embodiment 15 An embodiment of any one of embodiments 7-14, wherein at least one of the terminal hydrocarbon chains is alkyl.
  • Embodiment 16 An embodiment of any one of embodiments 7-15, wherein at least one of the terminal hydrocarbon chains is alkenoxy.
  • Embodiment 17 An embodiment of any one of embodiments 1-6, wherein the permanently charged cationic lipid comprises: one or more quaternary ammonium nitrogens; two or more carbon branch points each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkoxy, alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members; and three or more terminal hydrocarbon chains not directly bonded to the one or more quaternary ammonium nitrogens, wherein the terminal hydrocarbon chains are each independently alkyl or alkenyl.
  • Embodiment 18 An embodiment of embodiment 17, wherein the two or more carbon branch points are each independently directly bonded to three or more optionally oxo- substituted and optionally halogen-substituted alkyl, alkoxy, alkenoxy, heteroalkyl, or heteroalkenyl groups.
  • Embodiment 19 An embodiment of embodiment 17 or 18, wherein the two or more carbon branch points are each independently directly bonded to at least one optionally oxo- substituted and optionally halogen-substituted alkyl group.
  • Embodiment 20 An embodiment of any one of embodiments 17-19, wherein at least one of the two or more carbon branch points is directly bonded to at least two optionally oxo- substituted and optionally halogen-substituted alkyl groups.
  • Embodiment 21 An embodiment of any one of embodiments 17-20, wherein at least one of the terminal hydrocarbon chains is alkyl.
  • Embodiment 22 An embodiment of any one of embodiments 17-21, wherein at least one of the terminal hydrocarbon chains is alkenyl.
  • Embodiment 23 An embodiment of any one of embodiments 1-6, wherein the permanently charged cationic lipid comprises: one or more quaternary ammonium nitrogens; and one or more hydrolyzable linkages, wherein each of the one or more hydrolyzable linkages is independently a silyl ether linkage, a siloxane linkage, a disulfide linkage, a carbonate linkage, a pyrrolidine dicarboxylate linkage, or a cyclic benzylidene acetal linkage.
  • Embodiment 24 An embodiment of embodiment 23, wherein at least one of the one or more hydrolyzable linkages is a silyl ether linkage.
  • Embodiment 25 An embodiment of embodiment 23, wherein each of the one or more hydrolyzable linkages is a silyl ether linkage.
  • Embodiment 26 An embodiment of any one of embodiments 1-25, wherein the molar ratio of the ionizable lipid or the pharmaceutically acceptable salt thereof to the permanently charged cationic lipid or the pharmaceutically acceptable salt thereof is between about 2:1 and about 60:1.
  • Embodiment 27 An embodiment of any one of embodiments 1-26, wherein the permanently charged cationic lipid or the pharmaceutically acceptable salt thereof comprises between 0.5 mol% and 30 mol% of the total lipid of the lipid nanoparticle.
  • Embodiment 28 An embodiment of any one of embodiments 1-27, wherein the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 30 mol% and 70 mol% of the total lipid of the lipid nanoparticle.
  • Embodiment 29 An embodiment of any one of embodiments 1-28, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises between 1 mol% and 20 mol% of the total lipid of the lipid nanoparticle.
  • Embodiment 30 An embodiment of any one of embodiments 1-29, wherein the cholesterol or the derivative thereof comprises between 20 mol% and 60 mol% of the total lipid of the lipid nanoparticle.
  • Embodiment 31 An embodiment of any one of embodiments 1-30, wherein the conjugated lipid comprises a polyethylene glycol (PEG)-lipid conjugate.
  • Embodiment 32 An embodiment of any one of embodiments 1-31, wherein the conjugated lipid comprises between 0.1 mol% and 5 mol% of the total lipid of the lipid nanoparticle.
  • Embodiment 33 An embodiment of any one of embodiments 1-32, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 39 mol% and 49 mol% of the total lipid of the lipid nanoparticle; the phospholipid or the pharmaceutically acceptable salt thereof comprises between 8 mol% and 12 mol% of the total lipid of the lipid nanoparticle; the cholesterol or the derivative thereof comprises between 33 mol% and 43 mol% of the total lipid of the lipid nanoparticle; the conjugated lipid comprising between 0.5 mol% and 3 mol% of the total lipid of the lipid nanoparticle; and the permanently charged cationic lipid or the pharmaceutically acceptable salt thereof comprises between 1 mol% and 11 mol% of the total lipid of the lipid nanoparticle.
  • Embodiment 34 An embodiment of any one of embodiments 1-32, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 39 mol% and 49 mol% of the total lipid of the lipid nanoparticle; the phospholipid or the pharmaceutically acceptable salt thereof comprises between 9 mol% and 11 mol% of the total lipid of the lipid nanoparticle; the cholesterol or the derivative thereof comprises between 37 mol% and 40 mol% of the total lipid of the lipid nanoparticle; the conjugated lipid comprising between 1 mol% and 3 mol% of the total lipid of the lipid nanoparticle; and the permanently charged
  • Embodiment 35 An embodiment of any one of embodiments 1-34, wherein the lipid nanoparticle has a pKa that is no less than 6.4.
  • Embodiment 36 An embodiment of embodiment 35, wherein the lipid nanoparticle has a pKa between 6.4 and 6.9.
  • Embodiment 37 An embodiment of embodiment 36, wherein the lipid nanoparticle has a pKa between 6.4 and 6.7.
  • Embodiment 38 An embodiment of embodiment 37, wherein the lipid nanoparticle has a pKa between 6.4 and 6.6.
  • Embodiment 39 An embodiment of embodiment 38, wherein the lipid nanoparticle has a pKa between 6.4 and 6.5.
  • Embodiment 40 An embodiment of embodiment 37, wherein the lipid nanoparticle has a pKa between 6.5 and 6.7.
  • Embodiment 41 An embodiment of embodiment 40, wherein the lipid nanoparticle has a pKa between 6.5 and 6.6.
  • Embodiment 42 An embodiment of embodiment 40, wherein the lipid nanoparticle has a pKa between 6.6 and 6.7.
  • Embodiment 43 A lipid nanoparticle comprising: a nucleic acid; an ionizable lipid or a pharmaceutically acceptable salt thereof; a phospholipid or a pharmaceutically acceptable salt thereof; cholesterol or a derivative thereof; a conjugated lipid; and a permanently charged cationic lipid or a pharmaceutically acceptable salt thereof, the permanently charged cationic lipid comprising: one or more quaternary ammonium nitrogens; a heteroatom branch point other than the one or more quaternary ammonium nitrogens, the heteroatom branch point directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkoxy, alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members; and three or more terminal hydrocarbon chains not directly bonded to the one or more quaternary ammonium nitrogens, wherein the terminal hydrocarbon chains are each independently alkyl or alkenyl.
  • Embodiment 44 An embodiment of embodiment 43, wherein the heteroatom branch point is a silicon.
  • Embodiment 45 An embodiment of embodiment 43, wherein the heteroatom branch point is a nitrogen.
  • Embodiment 46 An embodiment of any one of embodiments 43-45, wherein the heteroatom branch point is directly bonded to at least one optionally oxo-substituted and optionally halogen-substituted heteroalkenyl group having five or more members.
  • Embodiment 47 An embodiment of any one of embodiments 43-46, wherein the heteroatom branch point is directly bonded to at least one optionally oxo-substituted and optionally halogen-substituted alkenoxy group having five or more members.
  • Embodiment 48 An embodiment of embodiment 47, wherein the heteroatom branch point is directly bonded to at least two optionally oxo-substituted and optionally halogen- substituted alkenoxy groups, each independently having five or more members.
  • Embodiment 49 An embodiment of any one of embodiments 43-48, wherein the heteroatom branch point is directly bonded to at least one optionally oxo-substituted and optionally halogen-substituted alkyl group having five or more members.
  • Embodiment 50 An embodiment of embodiment 49, wherein the heteroatom branch point is directly bonded to at least two optionally oxo-substituted and optionally halogen- substituted alkyl groups, each independently having five or more members.
  • Embodiment 51 An embodiment of any one of embodiments 43-50, wherein at least one of the terminal hydrocarbon chains is alkyl.
  • Embodiment 52 An embodiment of any one of embodiments 43-51, wherein at least one of the terminal hydrocarbon chains is alkenyl.
  • Embodiment 53 A lipid nanoparticle comprising: a nucleic acid; an ionizable lipid or a pharmaceutically acceptable salt thereof; a phospholipid or a pharmaceutically acceptable salt thereof; cholesterol or a derivative thereof; a conjugated lipid; and a permanently charged cationic lipid or a pharmaceutically acceptable salt thereof, the permanently charged cationic lipid comprising: one or more quaternary ammonium nitrogens; two or more carbon branch points, each independently directly bonded to three or more optionally oxo-substituted and optionally halogen-substituted alkyl, alkenyl, alkoxy, alkenoxy, heteroalkyl, or heteroalkenyl
  • Embodiment 54 An embodiment of embodiment 53, wherein the two or more carbon branch points are each independently directly bonded to three or more optionally oxo- substituted and optionally halogen-substituted alkyl, alkoxy, alkenoxy, heteroalkyl, or heteroalkenyl groups, each independently having five or more members.
  • Embodiment 55 An embodiment of any one of embodiments 53 or 54, wherein the two or more carbon branch points are each independently directly bonded to at least one optionally oxo-substituted and optionally halogen-substituted alkyl group having five or more members.
  • Embodiment 56 An embodiment of any one of embodiments 53-55, wherein at least one of the two or more carbon branch points is directly bonded to at least two optionally oxo- substituted and optionally halogen-substituted alkyl groups, each independently having five or more members.
  • Embodiment 57 An embodiment of any one of embodiments 53-56, wherein at least one of the terminal hydrocarbon chains is alkyl.
  • Embodiment 58 An embodiment of any one of embodiments 53-57, wherein at least one of the terminal hydrocarbon chains is alkenyl.
  • Embodiment 59 A lipid nanoparticle comprising: a nucleic acid; an ionizable lipid or a pharmaceutically acceptable salt thereof; a phospholipid or a pharmaceutically acceptable salt thereof; cholesterol or a derivative thereof; a conjugated lipid; and a permanently charged cationic lipid or a pharmaceutically acceptable salt thereof, the permanently charged cationic lipid comprising: one or more quaternary ammonium nitrogens; and one or more hydrolyzable linkages, wherein each of the one or more hydrolyzable linkages is independently a silyl ether linkage, a siloxane linkage, a disulfide linkage, a carbonate linkage, a pyrrolidine dicarboxylate linkage, or a cyclic benzylidene acetal linkage.
  • Embodiment 59 A lipid nanop
  • Embodiment 61 An embodiment of embodiment 59, wherein each of the one or more hydrolyzable linkages is a silyl ether linkage.
  • Embodiment 62 An embodiment of any one of embodiments 43-61, wherein the molar ratio of the ionizable lipid or the pharmaceutically acceptable salt thereof to the permanently charged cationic lipid or the pharmaceutically acceptable salt thereof is between about 2:1 and about 60:1.
  • Embodiment 63 An embodiment of any one of embodiments 43-62, wherein the permanently charged cationic lipid or the pharmaceutically acceptable salt thereof comprises between 0.5 mol% and 30 mol% of the total lipid of the lipid nanoparticle.
  • Embodiment 64 An embodiment of any one of embodiments 43-63, wherein the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 30 mol% and 70 mol% of the total lipid of the lipid nanoparticle.
  • Embodiment 65 An embodiment of any one of embodiments 43-64, wherein the phospholipid or the pharmaceutically acceptable salt thereof comprises between 1 mol% and 20 mol% of the total lipid of the lipid nanoparticle.
  • Embodiment 66 An embodiment of any one of embodiments 43-65, wherein the cholesterol or the derivative thereof comprises between 20 mol% and 60 mol% of the total lipid of the lipid nanoparticle.
  • Embodiment 67 An embodiment of any one of embodiments 43-66, wherein the conjugated lipid comprises a PEG-lipid conjugate.
  • Embodiment 68 An embodiment of any one of embodiments 43-67, wherein the conjugated lipid comprises between 0.1 mol% and 5 mol% of the total lipid of the lipid nanoparticle.
  • Embodiment 69 An embodiment of any one of embodiments 43-68, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 39 mol% and 49 mol% of the total lipid of the lipid nanoparticle; the phospholipid or the pharmaceutically acceptable salt thereof comprises between 8 mol% and 12 mol% of the total lipid of the lipid nanoparticle; the cholesterol or the derivative thereof comprises between 33 mol% and 43 mol% of the total lipid of the lipid nanoparticle; the conjugated lipid comprising between 0.5 mol% and 3 mol% of the total lipid of the lipid nanoparticle; and the permanently charged
  • Embodiment 70 An embodiment of any one of embodiments 43-68, wherein: the ionizable lipid or the pharmaceutically acceptable salt thereof comprises between 39 mol% and 49 mol% of the total lipid of the lipid nanoparticle; the phospholipid or the pharmaceutically acceptable salt thereof comprises between 9 mol% and 11 mol% of the total lipid of the lipid nanoparticle; the cholesterol or the derivative thereof comprises between 37 mol% and 40 mol% of the total lipid of the lipid nanoparticle; the conjugated lipid comprising between 1 mol% and 3 mol% of the total lipid of the lipid nanoparticle; and the permanently charged cationic lipid or the pharmaceutically acceptable salt thereof comprises between 1 mol% and 11 mol% of the total lipid of the lipid nanoparticle.
  • Embodiment 71 An embodiment of any one of embodiments 43-70, wherein the nucleic acid comprises RNA.
  • Embodiment 72 An embodiment of embodiment 71, wherein the RNA comprises siRNA, miRNA, shRNA, asRNA, DsiRNA, piRNA, or a combination thereof.
  • Embodiment 73 An embodiment of embodiment 72, wherein the RNA comprises siRNA.
  • Embodiment 74 An embodiment of any one of embodiments 43-70, wherein the nucleic acid comprises an antisense oligonucleotide, a morpholino oligonucleotide, or a combination thereof.
  • Embodiment 75 An embodiment of any one of embodiments 43-74, wherein the lipid nanoparticle has a pKa that is no less than 6.4.
  • Embodiment 76 An embodiment of embodiment 75, wherein the lipid nanoparticle has a pKa between 6.4 and 6.9.
  • Embodiment 77 An embodiment of embodiment 76, wherein the lipid nanoparticle has a pKa between 6.4 and 6.7.
  • Embodiment 78 An embodiment of embodiment 77, wherein the lipid nanoparticle has a pKa between 6.4 and 6.6.
  • Embodiment 79 An embodiment of embodiment 78, wherein the lipid nanoparticle has a pKa between 6.4 and 6.5.
  • Embodiment 80 An embodiment of embodiment 77, wherein the lipid nanoparticle has a pKa between 6.5 and 6.7.
  • Embodiment 81 An embodiment of embodiment 80, wherein the lipid nanoparticle has a pKa between 6.5 and 6.6.
  • Embodiment 82 An embodiment of embodiment 80, wherein the lipid nanoparticle has a pKa between 6.6 and 6.7.
  • Embodiment 83 An embodiment of any one of embodiments 43-82, wherein the nucleic acid is configured to reduce expression of a gene in a hepatic stellate cell.
  • Embodiment 84 An embodiment of embodiment 83, wherein the gene encodes COL1A1, COL1A2, COL2A1, COL3A1, COL4A1, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL7A1, COL8A2, COL9A1, COL9A2, COL9A3, COL10A1, COL11A1, COL11A2, COL17A1, COL18A1, TIMP1, MMP2, MMP1, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP22, MMP23, MMP24, MMP25, MMP26, MMP28, ⁇ -SMA, CTGF, HSP47, LOX, EGFR, ROCK, ⁇ -S
  • Embodiment 85 A pharmaceutical composition comprising: the lipid nanoparticle of any one of embodiments 43-84; and a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient.
  • Embodiment 86 An embodiment of embodiment 83 or 84 for use in reducing expression of the gene in the hepatic stellate cell.
  • Embodiment 87 The use of the lipid nanoparticle of 83 or 84 to prepare a medicament for reducing expression of the gene in the hepatic stellate cell.
  • Embodiment 88 A method for the in vivo delivery of the nucleic acid of any one of embodiments 43-84, the method comprising administering to a subject the lipid nanoparticle of any one of embodiments 43-84 or the pharmaceutical composition of embodiment 85.
  • Embodiment 89 A lipid nanoparticle of any one of embodiments 43-84, or pharmaceutical composition of claim 85, for use in the in vivo delivery of the nucleic acid of any one of claims 43-84 to a subject.
  • Embodiment 90 The use of the lipid nanoparticle of any one of embodiments 43-84, or the pharmaceutical composition of embodiment 85, to prepare a medicament for the in vivo delivery of the nucleic acid of any one of embodiments 43-84 to a subject.
  • Embodiment 91 A method for preventing or treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the lipid nanoparticle of any one of any one of embodiments 43-84, or the pharmaceutical composition of embodiment 85.
  • Embodiment 92 An embodiment of embodiment 91, wherein the disease or disorder comprises an infection, a liver disease or disorder, or a cancer.
  • Embodiment 93 An embodiment of embodiment 92, wherein the liver disease or disorder comprises a disease or disorder associated with hepatic stellate cells.
  • Embodiment 94 An embodiment of embodiment 92 or 93, wherein the disease or disorder comprises liver fibrosis, liver cirrhosis, hepatocellular carcinoma (HCC), nonalcoholic fatty liver disease (NASH), nonalcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), alcoholic liver disease (ALD), immune-induced hepatitis, ischemia and reperfusion (I/R) liver injury, schistosome viral hepatitis, or chronic viral hepatitis.
  • HCC hepatocellular carcinoma
  • NASH nonalcoholic fatty liver disease
  • NAFLD nonalcoholic fatty liver disease
  • ALD alcoholic liver disease
  • I/R immune-induced hepatitis
  • I/R immune-induced hepatitis
  • I/R immune-induced hepatitis
  • I/R immune
  • Embodiment 95 A lipid nanoparticle of any one of embodiments 43-84, or pharmaceutical composition of embodiment 85, for use in preventing or treating a disease or disorder in a subject.
  • Embodiment 96 The lipid nanoparticle or pharmaceutical composition of embodiment 95, wherein the disease or disorder comprises an infection, a liver disease or disorder, or a cancer.
  • Embodiment 97 The lipid nanoparticle or pharmaceutical composition of embodiment 96, wherein the liver disease or disorder comprises a disease or disorder associated with hepatic stellate cells.
  • Embodiment 98 The lipid nanoparticle or pharmaceutical composition of embodiment 96 or 97, wherein the disease or disorder comprises liver fibrosis, liver cirrhosis, HCC, NASH, NAFLD, NAFL, ALD, immune-induced hepatitis, I/R liver injury, schistosome viral hepatitis, or chronic viral hepatitis.
  • Embodiment 99 The use of a lipid nanoparticle of any one of embodiments 43-84, or pharmaceutical composition of embodiment 85, to prepare a medicament for preventing or treating a disease or disorder in a subject.
  • Embodiment 100 An embodiment of embodiment 99, wherein the disease or disorder comprises an infection, a liver disease or disorder, or a cancer.
  • Embodiment 101 An embodiment of embodiment 100, wherein the liver disease or disorder comprises a disease or disorder associated with hepatic stellate cells.
  • Embodiment 102 An embodiment of embodiment 100 or 101, wherein the disease or disorder comprises liver fibrosis, liver cirrhosis, HCC, NASH, NAFLD, NAFL, ALD, immune-induced hepatitis, I/R liver injury, schistosome viral hepatitis, or chronic viral hepatitis.
  • the disease or disorder comprises liver fibrosis, liver cirrhosis, HCC, NASH, NAFLD, NAFL, ALD, immune-induced hepatitis, I/R liver injury, schistosome viral hepatitis, or chronic viral hepatitis.
  • EXAMPLES [0378] The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the disclosure in any manner. Example 1.
  • the permanently charged cationic lipids of the provided lipid nanoparticles can be synthesized by a variety of methods known to one of skill in the art (see, e.g., Comprehensive Organic Transformations, R.C. Larock, 1989) or by an appropriate combination of generally well known synthetic methods. Techniques useful in synthesizing the disclosed compounds are both readily apparent and accessible to those of skill in the relevant art.
  • the examples below are offered to illustrate certain of the diverse methods available for use in assembling the provided compounds. However, the examples are not intended to define the scope of reactions or reaction sequences that are useful in preparing the compounds. Exemplary structures below are named according to standard IUPAC nomenclature using the CambridgeSoft ChemDraw naming package.
  • Lipid X certain permanently charged cationic lipids and quaternary salts thereof are referred to as “Lipid X,” with X being a number, and certain (other) compounds (synthetic intermediates and reagents) are referred to as “Compound Y” or “cmpd. Y,” with Y being a number.
  • Pentadecan-8-yl 8- oxooctadecanoate (27) (4 g, 7.9 mmol was added and the reaction warmed 45 °C and stirred for 16 h. The reaction was quenched (H2O), extracted with Et2O, washed with brine, dried (MgSO4), filtered and concentrated in vacuo. The residue was purified by flash chromatography (0-4 % EtOAc/Hex) to give 1-tert-butyl 10-pentadecan-8-yl (2E)-3-decyldec- 2-enedioate (28) (4.53 g, 94.9 %). 6.
  • N-[3,4-Bis(methoxycarbonyl)pyrrolidin-1-yl]-2,2,2-trifluoroethanehydrogenio carboxylic acid (6.15 g, 20.4 mmol), Na2CO3 (1.19 g, 11.2 mmol) and di-tert-butyl dicarbonate (BOC anhydride) (5.4 g, 24.5 mmol) were stirred in acetone (84.3 mL) and H 2 O (22.14 mL) at RT for 3 h.
  • the organic solvent was removed under reduced pressure and the remaining aqueous residue poured into DCM and 0.5 M HCl and extracted with DCM.
  • Lipid 3 was prepared using General Synthetic Protocol 2 with precursor compound (9) and iodomethane.
  • Lipid 6 was prepared using General Synthetic Protocol 2 with precursor compound (9) and bromoethane modified to heat at 80 °C in MeCN.
  • Lipid 7 was prepared using General Synthetic Protocol 2 with precursor compound (9) and bromoethane modified to heat at 80°C in MeCN.
  • Lipid 12 was prepared using General Synthetic Protocol 2 with precursor compound (6) and bromoethane modified to heat at 80 °C in MeCN.
  • Lipid 14 was prepared using General Synthetic Protocol 2 with precursor compound (18) and iodomethane.
  • Lipid 15 was prepared from Lipid 14 by taking up in EtOAc, washing with 3 M HCl x 3 and 2 x brine (NaCl), drying (MgSO4), filtering, concentrating and purifying by flash chromatography (0-40% MeOH/DCM) to give the chloride quaternary salt.
  • Lipid 16 was prepared using General Synthetic Protocol 2 with precursor compound (19) and iodomethane.
  • Lipid 19 was prepared in analogous to Lipid 15 using 3 M HBr and NaBr to convert the iodide quaternary ammonium salt into the bromide.
  • the iodide precursor was prepared using General Synthetic Protocol 2 with precursor compound (36) and iodomethane.
  • Lipid 20 was prepared using General Synthetic Protocol 2 with precursor compound (49) and iodomethane.
  • Lipid 21 was prepared using General Synthetic Protocol 2 with precursor compound (37) and iodomethane.
  • Lipid 24 was prepared using General Synthetic Protocol 2 with precursor compound (34) and iodomethane.
  • Lipid 29 was prepared using General Synthetic Protocol 2 with precursor compound (93) and iodomethane.
  • Lipid nanoparticles were prepared from lipid ethanolic stock solutions containing five components: a PEG-conjugated lipid, an ionizable silyl ester lipid (N 1 ,N 3 -bis(4-(bis(((Z)-dec- 4-en-1-yl)oxy)(methyl)silyl)butyl)-N 1 ,N 3 -dimethylpropane-1,3-diamine), a quaternary salt, cholesterol, and a phospholipid (e.g., DSPC) in the molar ratios as listed in Table 2.
  • the combined lipid stocks had a concentration of approximately 8-8.5 mg/mL total lipid in 100% ethanol.
  • lipid and nucleic acid solutions were blended at a flow rate of 400 mL/min through a T-connector into ⁇ 4 volumes of phosphate buffered saline (PBS), pH 7.4.
  • PBS phosphate buffered saline
  • Formulations were placed in Slide-A-Lyzer dialysis units (ThermoFisher, MWCO 10,000) and dialyzed overnight against 10 mM Tris, 500 mM NaCl, pH 8 buffer. Following dialysis, the formulations were concentrated using VivaSpin concentrator units (Cytiva, MWCO 100,000) and dialyzed overnight against 5 mM Tris, 10%
  • Comparative lipid nanoparticles were prepared from lipid solutions containing four components: a PEG-conjugated lipid, an ionizable lipid, cholesterol, and a phospholipid (e.g., DSPC), at a molar ratio of about 1.5:50:38.5:10. These lipid nanoparticles, denoted as Composition 1 in Table 2, were otherwise prepared following the procedures described above for the five-component lipid nanoparticles.
  • Composition 1 in Table 2 were otherwise prepared following the procedures described above for the five-component lipid nanoparticles.
  • LNP formulations encapsulating RELN (a hepatic stellate cell gene target) siRNA were injected intravenously at 0.025 mg/kg to female BALB/c mice (6-8 weeks old). On the day of injection, the LNP stocks were thawed, filtered, and diluted to the required dosing concentration with PBS. pH 7.4. At 48 hours post-dose the mice were anaesthetized with a lethal dosage of ketamine/xylazine. Liver samples (left lateral lobe) were collected into
  • RNAlater TM Stabilization Solution 145 78645288V.1 RNAlater TM Stabilization Solution (Invitrogen, AM7024) and stored at 2-8 °C until analysis. Knockdown of RELN mRNA was assessed by QuantiGene assay (ThermoFisher) following the manufacturer’s instructions, with RELN mRNA expression normalized to the housekeeping gene GAPDH. A decrease in the relative mRNA signal (RELN:GAPDH) compared to that of the PBS-treated group indicates an increase in % reelin knockdown and improved gene silencing in hepatic stellate cells. [0447] Table 4 presents results from experiments showing the RELN:GAPDH mRNA ratios observed following administration of LNP compositions from Table 2 or PBS controls.
  • Composition 5 LNP was formulated with RELN siRNA as described in Example 2 with some minor changes: the starting lipid stock and nucleic acid concentrations were doubled, and LNP was diluted with an additional 1.4x volumes of PBS. The resulting formulation was processed by tangential flow ultrafiltration into the cryobuffer before being sterile filtered, aliquoted, and frozen at -80 °C.
  • LNP was dosed as in Example 3 to male Sprague-Dawley rats for four weekly doses at 0.1, 1.0, 3.0 or 6.0 mg/kg.
  • RNAlater TM Liver enzyme levels were assessed 24 hours after the last dose with IDEXX (Delta, BC). Livers were collected into RNAlater TM for QuantiGene analysis of RELN mRNA knockdown. [0450] Composition 5 reached maximal RELN gene knockdown at 1 mg/kg (FIG. 1) and resulted in no increase in alanine transaminase (ALT) and aspartate aminotransferase (AST)
  • Example 5 Effect of quaternary salt on siRNA LNP gene silencing activity in hepatic stellate cells
  • Lipid nanoparticles were prepared as described in Example 2, but with formulations having different quaternary salts or other positively charged cationic lipids (e.g., DOTAP), and different ionizable lipids, as shown in Table 5. Particle characteristics for these LNP formulations were determined as in Example 2, with the resulting particle size (Z-average), polydispersity index (PDI), and percent encapsulation values presented in Table 6.
  • Lipid nanoparticles were prepared as described in Example 2, but with formulations having different parent ionizable lipids that do not contain silicon, and their derivative quaternary salts as shown in Table 8. Particle characteristics for these LNP formulations were determined as in Example 2, with the resulting particle size (Z-average), polydispersity index (PDI), and percent encapsulation values presented in Table 9. Gene silencing activity assessment of the lipid nanoparticles was performed as described in Example 3, with the results
  • Lipid nanoparticles were prepared according to Composition 1 or 5 as described in Example 2, but with some populations of the prepared LNPs encapsulating siRNA targeting the hepatic stellate cell gene RELN, and other populations encapsulating siRNA targeting the hepatocyte gene TTR.
  • the LNP formulations were injected intravenously into female BALB/c mice (6–8 weeks old) at doses ranging from 0.001 to 0.025 mg/kg. LNP administration, liver sample collection, and mRNA knockdown assessments were otherwise carried out as described in Example 3.
  • FIG. 5 presents results showing that administration of Composition 5 LNPs formulated with quaternary salts and encapsulating RELN siRNA resulted in a significantly higher percent knockdown of RELN mRNA in whole liver lysates than administration of Composition 1 LNPs that also encapsulated RELN siRNA but that were formulated without quaternary salts. This indicates that the presence of the quaternary salt lipid increased delivery of the siRNA to hepatic stellate cells.
  • FIG. 5 presents results showing that administration of Composition 5 LNPs formulated with quaternary salts and encapsulating TTR siRNA resulted in a significantly lower percent knockdown of TTR mRNA in whole liver
  • LNP pKa Determination of LNP pKa
  • TNS 6-(p-toluidino)-2-naphthalenesulfonyl chloride
  • LNPs were diluted to 40 ⁇ M total lipid and TNS was diluted to 6 ⁇ M in buffered solutions containing 25 mM citric acid, 20 mM ammonium acetate, and 150 mM NaCl, and having pH values ranging from 5.0 to 8.7.
  • the samples were incubated at 37 °C and fluorescence was measured with an excitation wavelength of 322 nm and an emission wavelength of 431 nm.
  • the pKa was determined by plotting fluorescence vs. pH and assigning the LNP pKa as the pH where fluorescence was equal to 50% of total fluorescence.
  • the pKa of LNPs formulated with siRELN without quaternary salt was determined to be 6.1.

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Abstract

La présente divulgation concerne des nanoparticules lipidiques qui sont avantageusement efficaces dans le ciblage de cellules hépatiques étoilées avec une spécificité améliorée. Les nanoparticules lipidiques comprennent cinq composants lipidiques, comprenant un lipide cationique chargé en permanence qui est un lipide d'ammonium quaternaire. La divulgation concerne également des compositions pharmaceutiques et des procédés comprenant les nanoparticules lipidiques selon l'invention.
PCT/IB2025/057322 2024-07-22 2025-07-19 Formulations de particules lipidiques contenant des lipides cationiques chargés en permanence Pending WO2026022656A2 (fr)

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