PEG lipids and lipid nanoparticles

Abstract

The present invention relates to compounds of formula (I): [Formula 1] TIFF2024500673000026.tif28165 or a salt thereof (wherein, R 1 , n, L and X have any of the values ​​described herein), and compositions and lipid nanoparticles comprising the compounds of formula I, and uses thereof for delivering therapeutic agents such as siRNA and mRNA.

Description

Cross-reference of related applications

[0001] This application claims priority to U.S. Patent Application No. 63 / 127,684, filed on 18 December 2020, which is incorporated herein by reference. [Background technology]

[0002] Lipid nanoparticles (LNPs) are effective drug delivery systems for biologically active compounds that are normally impermeable to cells, such as therapeutic nucleic acids, proteins, and peptides. For example, nucleic acid-based drugs, including large nucleic acid molecules such as in vitro transcription messenger RNA (mRNA) and smaller polynucleotides that interact with messenger RNA or genes, must be delivered to the appropriate cellular compartment to be effective. Double-stranded nucleic acids, such as double-stranded RNA molecules (dsRNA) (including siRNA), have physicochemical properties that make them impermeable to cells. Once delivered to the appropriate compartment, siRNA blocks gene expression through a highly conserved regulatory mechanism known as RNA interference (RNAi). Typically, siRNA is large in size, ranging from 12 to 17 kDa, and highly anionic due to its phosphate backbone with up to 50 negative charges. Furthermore, the two complementary RNA strands result in a rigid helix. These characteristics contribute to the poor "drug-like" properties of siRNA. When administered intravenously, siRNA is rapidly eliminated from the body with a typical half-life of only 10 minutes. Furthermore, siRNA has been shown to be rapidly degraded by nucleases present in the blood and other bodily fluids or tissues, stimulating a potent immune response in vitro and in vivo. See, for example, Non-Patent Document 1. mRNA molecules have similar problems with impermeability, fragility, and immunogenicity. See Patent Document 1.

[0003] Lipid nanoparticle formulations have improved nucleic acid delivery in vivo. For example, such formulations can significantly reduce the siRNA dose required to achieve targeted knockdown in vivo. See Non-Patent Literature 2. Typically, such lipid nanoparticle drug delivery systems are multi-component formulations comprising cationic (or ionizable) lipids, helper lipids, and lipids containing polyethylene glycol. Note that the terms “cationic” and “ionizable” in this specification are used interchangeably with respect to lipids unless otherwise specified. Positively charged cationic lipids bind to anionic nucleic acids, while other components assist in the stable self-assembly of lipid nanoparticles.

[0004] Efforts are being made to improve the delivery efficacy of lipid nanoparticle formulations. Many of these efforts are directed towards developing more suitable cationic lipids. See, for example, Non-Patent Documents 3-6. Furthermore, the PEG lipid PEG2000-C-DMA is used in LNP formulations that are in human clinical trials for diverse applications such as oncology, vaccines, antivirals, and metabolic diseases. These LNP formulations are used to deliver therapeutic payloads containing, but not limited to, oligonucleotides such as plasmid DNA, siRNA, mRNA, and self-replicating RNA. Despite these efforts, there is still a need for lipid nanoparticles containing formulations that provide high efficacy after administration and enable the delivery of low doses of nucleic acids, such as mRNA and siRNA. For example, there is a need for additional PEG lipids with, for example, unique or improved properties for use in lipid nanoparticle formulations. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2016 / 118697 [Non-patent literature]

[0006] [Non-Patent Document 1] Robbins et al., Oligonucleotides 19:89-102, 2009 [Non-Patent Document 2] Zimmermann et al., Nature 441:111-114, 2006 [Non-Patent Document 3] Akinc et al., Nature Biotechnology 26:561-569, 2008 [Non-Patent Document 4] Love et al., Proc. Natl. Acad. Sci. USA 107:1864-1869, 2010 [Non-Patent Document 5] Baigude et al., Journal of Controlled Release 107:276-287, 2005 [Non-Patent Document 6] Semple et al., Nature Biotechnology 28:172-176, 2010 [Overview of the project] [Means for solving the problem]

[0007] Therefore, in this specification, compounds of formula (I): [ka] or its salt (in the formula, R 1 is H, (C1-C6) alkyl, or (C1-C6) alkanoyl, n is an integer in the range of approximately 10 to approximately 150, and L does not exist, and X is -C(=O)NR 2 R 3 is; or L is (C1-C6) alkyl, and X is -N(R 4 )C(=O)CH(R 2 )(R 3 ), -OCH(R 2 )(R3 ), -C(=O)OCH2CH(R 2 )(R 3 ), -N(R 4 )C(=O)N(R 2 )(R 3 ), and -SO2N(R 2 )(R 3 ) selected from the group consisting of; R 2 is (C 10 -C 20 ) alkyl; R 3 is (C 10 -C 20 ) alkyl; and R 4 is H or (C1-C6) alkyl) is provided.

[0008] [[ID=3&]] This compound can be used, for example, as a component of lipid nanoparticles useful for delivering therapeutic agents such as siRNA and mRNA.

[0009] As used herein, lipid nanoparticles and pharmaceutical compositions containing the lipid nanoparticles are provided. These lipid nanoparticles and pharmaceutical compositions are particularly useful for delivering nucleic acids to patients (e.g., humans) or cells.

[0010] The present invention also provides a pharmaceutical composition comprising the lipid nanoparticles of the present invention and a pharmaceutically acceptable carrier.

[0011] The present invention also provides a method for delivering nucleic acids to cells, the method comprising contacting the cells with the lipid nanoparticles of the present invention. More generally, the present invention provides a method for administering nucleic acids to living cells in vivo or in vitro. <ooo0355> The present invention also provides a method for treating a disease characterized by a deficiency of a functional protein (e.g., caused by a genetic deficiency), comprising administering the lipid nanoparticles of the present invention to a subject having the disease, wherein the nucleic acid molecule is a functional protein or mRNA encoding a protein having the same biological activity as the functional protein.

[0013] The present invention also provides a method for treating a disease characterized by the overexpression of a polypeptide, comprising administering the lipid nanoparticles of the present invention to a subject having the disease, wherein the nucleic acid molecule is an siRNA that targets the expression of the overexpressed polypeptide.

[0014] The present invention also provides lipid nanoparticles for therapeutic or prophylactic treatment of diseases characterized by a deficiency of a functional protein (e.g., a genetic deficiency).

[0015] The present invention also provides lipid nanoparticles for therapeutic or prophylactic treatment of diseases characterized by polypeptide overexpression.

[0016] The present invention also provides a method for treating a disease or disorder in an animal, comprising administering a therapeutically effective amount of the lipid nanoparticles of the present invention to the animal.

[0017] The present invention also provides processes and intermediates disclosed herein that are useful for producing the lipid nanoparticles of the present invention. [Brief explanation of the drawing]

[0018] [Figure 1] The results of the anti-PEG antibody analysis are shown, with the results for each compound displayed from left to right at day 0, day 7, and day 14. [Figure 2] The results of the anti-PEG antibody analysis are shown, with the results for each compound displayed from left to right at day 0, day 7, and day 14. [Figure 3]The results of the EPO expression analysis are shown, with the results for each compound displayed from left to right at day 0, day 7, and day 14. [Figure 4] The results of the lipid clearance analysis are shown. PEG-C-DMA is the intermediate trace at 90 minutes, compound 9 is the lower trace at 90 minutes, and compound 31 is the upper trace at 90 minutes. [Figure 5] The results of the OTC expression analysis are shown. [Figure 6] The results of the anti-OVA IgG analysis are shown, with the results for each compound displayed from left to right at day 0, day 7, day 14, day 21, day 28, and day 35. [Modes for carrying out the invention]

[0019] definition Unless otherwise specified, the following definitions shall be used: Halo or halogen refers to fluoro, chloro, bromo, or iodo. Alkyl refers to both linear and branched groups, but when referring to individual radicals such as propyl, it includes only linear radicals, while branched isomers such as isopropyl are specifically mentioned.

[0020] The term "alkyl" means, unless otherwise specified, a linear or branched hydrocarbon radical having the indicated number of carbon atoms (i.e., (C1-C8)alkyl means 1 to 8 carbon atoms), either alone or as part of another substituent. Examples include (C1-C8)alkyl, (C2-C8)alkyl, (C1-C6)alkyl, (C2-C6)alkyl, and (C3-C6)alkyl. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and their higher homologues and isomers.

[0021] When used herein, the wavy line intersecting the bond in the chemical structure refers to a wavy line. TIFF0007897238000002.tif12165 shows the points where wavy bonds intersect in the chemical structure and are added to the rest of the molecule.

[0022] The terms “treat,” “treatment,” or “treating” include, to the extent that they relate to a disease or condition, inhibiting a disease or condition, eliminating a disease or condition, and / or reducing one or more symptoms of a disease or condition. The terms “treat,” “treatment,” or “treating” also refer to both therapeutic measures and / or preventive measures or preventive actions, the purpose of which is to prevent or slow (mitigate) an undesirable physiological change or impairment, such as the onset or spread of cancer. Beneficial or desired clinical outcomes, for example, include, but are not limited to, symptom reduction, attenuation of the degree of disease or impairment, stabilization of the disease or impairment (i.e., no worsening), delay or slowing of disease progression, relief or temporary relief of the disease state or impairment, and remission (partial or complete), whether detectable or undetectable. “Treat,” “treatment,” or “treating” can also mean extending survival compared to the predicted survival if no treatment is received. Those who require treatment include individuals who already have a disease or disability, those who are prone to developing a disease or disability, or those who need to prevent a disease or disability. In one embodiment, “treat,” “treatment,” or “treating” does not include “preventing” or “prevention.”

[0023] The terms “therapeutic dose” or “effective dose” include, but are not limited to, an amount of a compound that (i) treats or prevents a particular disease, condition or disorder; (ii) reduces, improves or eliminates one or more symptoms of a particular disease, condition or disorder; or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition or disorder as described herein.

[0024] As used herein, the term “mammal” refers to humans, higher non-human primates, rodents, domestic animals, cattle, horses, pigs, sheep, dogs, and cats. In one embodiment, the mammal is a human. As used herein, the term “patient” refers to any animal, including mammals. In one embodiment, the patient is a mammalian patient. In one embodiment, the patient is a human patient.

[0025] The compounds disclosed herein may also exist as tautomers in certain cases. Although only one delocalized resonance structure is illustrated, all such forms are considered to be within the scope of the present invention.

[0026] The present invention is also limited to, but does not limit, deuterium ( 2 It will be understood by those skilled in the art that the claimed compounds include any compound that can be enriched in any or all atoms beyond the naturally occurring isotopic ratios in one or more isotopes such as H or D). In a non-limiting example, the -CH3 group may be substituted with -CD3.

[0027] The pharmaceutical compositions of the present invention may contain one or more excipients. When used in combination with the pharmaceutical compositions of the present invention, the term “excipient” generally refers to an additional component that, when combined with a compound of formula (I) or a pharmaceutically acceptable salt thereof, provides the corresponding composition. For example, when used in combination with the pharmaceutical compositions of the present invention, the term “excipient” includes, but is not limited to, carriers, binders, disintegrants, lubricants, sweeteners, fragrances, coatings, preservatives, and colorants.

[0028] The stereochemical definitions and conventions used herein generally follow those of SP. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York, and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., New York, 1994. Since the compounds of the present invention may contain asymmetric or chiral centers, they exist in a variety of stereoisomers. All stereoisomers of the compounds of the present invention (including, but not limited to, diastereomers, enantiomers, and atropisomers), as well as mixtures thereof (such as racemic mixtures), are intended to form part of the present invention. Many organic compounds exist as optically active compounds; that is, they have the ability to rotate the plane of polarization. When describing optically active compounds, the prefixes D and L, or R and S, are used to indicate the absolute configuration of the molecule around its chiral center(s). The prefixes d and l, or (+) and (-), are used to indicate that the compound rotates the plane polarization, with (-) or l meaning the compound is levorotatory. Compounds prefixed with (+) or d are dextrorotatory. In a given chemical structure, these stereoisomers are identical except when they are mirror images of each other. Special stereoisomers can also be called enantiomers, and mixtures of such isomers are often called enantiomer mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemic compound, and can be found when a chemical reaction or process lacks stereoselectivity or stereospecificity. The terms "racemic mixture" and "racemic compound" refer to an equimolar mixture of two enantiomer species that does not exhibit optical activity.

[0029] It will be apparent to those skilled in the art that the compounds of the present invention having a chiral center can exist as optically active and racemic forms, and can be isolated as optically active and racemic forms. Some compounds may exhibit crystalline polymorphism. It should be understood that the present invention includes any racemic, optically active, crystalline polymorph, or stereoisomer of the compounds of the present invention, or mixtures thereof, that have the useful properties described herein, and methods for preparing optically active compounds (e.g., by recrystallization techniques, resolution of racemic forms, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase) are well known in the art.

[0030] In the compound formulas herein, where bonds are depicted in a non-stereochemical form (e.g., planar), the atoms to which the bonds are attached include all stereochemical possibilities. In the compound formulas herein, where bonds are depicted in a defined stereochemical form (e.g., thick line, thick wedge, dashed line, or dashed wedge), the atoms to which the stereochemical bonds are attached should be understood to include a large number of the shown absolute stereoisomers, unless otherwise specified. In one embodiment, the compound may be at least 51% of the shown absolute stereoisomers. In another embodiment, the compound may be at least 60% of the shown absolute stereoisomers. In another embodiment, the compound may be at least 80% of the shown absolute stereoisomers. In another embodiment, the compound may be at least 90% of the shown absolute stereoisomers. In another embodiment, the compound may be at least 95% of the shown absolute stereoisomers. In another embodiment, the compound may be at least 99% of the shown absolute stereoisomers.

[0031] The term "residue" applied to residues of a compound refers to a compound that has been modified in some way that results in the creation of an open valence, and the site of that open valence becomes the open valence. An open valence can be created by removing one or more atoms from a compound (e.g., removal of a single atom such as hydrogen, or removal of two or more atoms such as a group of atoms including but not limited to amines, hydroxyls, methyls, amides (e.g., -C(=O)NH2) or acetyl groups). An open valence can also be created by chemically transforming a first functional group of a compound into a second functional group of the compound (e.g., reduction of a carbonyl group, substitution of a carbonyl group with an amine, etc.), followed by the removal of one or more atoms from the second functional group.

[0032] The specific values ​​listed below for radicals, substituents, and ranges are for illustrative purposes only; they do not exclude other defined values ​​or other values ​​within the defined ranges for radicals and substituents. It should be understood that two or more values ​​can be combined. Also, it should be understood that the values ​​listed below (or subsets thereof) can be excluded.

[0033] Specifically, the (C1-C6) alkyl group may be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, 3-pentyl, or hexyl; the (C1-C6) alkanoyl group may be acetyl, propanoyl, or butanoyl.

[0034] In certain compounds of formula (I), L is absent and X is -C(=O)NR 2 R 3 Compounds such as the compound of formula (Ia): [ka] That is the case.

[0035] In a specific compound of formula (I), L is (C1-C6)alkyl and X is -N(R 4 )C(=O)CH(R 2 )(R 3) a compound that is, for example, a compound of formula (Ib): [ka] That is the case.

[0036] In a specific compound of formula (I), L is (C1-C6) alkyl and X is -OCH(R 2 )(R 3 ) a compound, for example, a compound of formula (Ic): [ka] That is the case.

[0037] In a specific compound of formula (I), L is (C1-C6) alkyl and X is -C(=O)CH2CH(R 2 )(R 3 ) a compound that is, for example, a compound of formula (Ib): [ka] That is the case.

[0038] In a specific compound of formula (I), L is (C1-C6)alkyl and X is -N(R 4 )C(=O)N(R 2 )(R 3 Compounds that are ) such as the compound of formula (Ie): [ka] That is the case.

[0039] In a specific compound of formula (I), L is (C1-C6) alkyl and X is -SO2N(R 2 )(R 3 Compounds that are, for example, compounds of formula (If): [ka] That is the case.

[0040] R 1 The specific value of is H.

[0041] R 1 The specific value is (C1-C6) alkyl.

[0042] R 1 The specific value is methyl.

[0043] R 1 The specific value is (C1-C6) alkanoyl.

[0044] R 1 The specific value is CH3C(=O)-.

[0045] The specific value of n is an integer in the range of approximately 20 to 100.

[0046] The specific value of n is an integer in the range of approximately 20 to 70.

[0047] The specific value of n is an integer in the range of approximately 20 to 60.

[0048] The specific value of n is an integer in the range of approximately 20 to 50.

[0049] The specific value of n is an integer in the range of approximately 30 to 100.

[0050] The specific value of n is an integer in the range of approximately 40 to 100.

[0051] The specific value of n is an integer in the range of approximately 40 to 60.

[0052] The specific value of n is an integer in the range of approximately 40 to 50.

[0053] The specific value of n is an integer in the range of approximately 44 to 46.

[0054] In one specific embodiment, the base: [ka] It is PEG-2000.

[0055] R 2 The specific value is C 11 -alkyl, C 12 -alkyl, C 13 -alkyl, C 14 -alkyl, C 15 -alkyl, C 16 -alkyl, C 17 -alkyl, C 18 -alkyl, C 19 -alkyl, or C 20 -It is alkyl.

[0056] R 2 The specific value is C 12 -alkyl, C 13 -alkyl, C 14 -alkyl, C 15 -alkyl, C 16 -alkyl, C 17 -alkyl or C 18 -It is alkyl.

[0057] R 2 The specific value is C 12 -alkyl, C 14 -alkyl, or C 16 -It is alkyl.

[0058] R 2 The specific meaning is C 14 -It is alkyl.

[0059] R 3 The specific value is C 11 -alkyl, C 12 -alkyl, C 13 -alkyl, C 14 -alkyl, C 15 -alkyl, C 16 -alkyl, C 17 -alkyl, C 18 -alkyl, C 19 -alkyl, or C 20 -It is alkyl.

[0060] R 3 The specific value of is C 12 -alkyl, C 13 -alkyl, C 14 -alkyl, C 15 -alkyl, C 16 -alkyl, C 17 -alkyl or C 18 -alkyl.

[0061] R 3 The specific value of is C 12 -alkyl, C 14 -alkyl, or C 16 -alkyl.

[0062] R 3 The specific meaning of is C 14 -alkyl.

[0063] R 4 The specific value of is H.

[0064] R 4 The specific value of is (C1-C6) alkyl.

[0065] R 4 The specific value of is methyl.

[0066] The term "about" means ±5%, ±4%, ±3%, ±2%, or ±1%.

[0067] The terms "interfering RNA," "RNAi," or "interfering RNA sequence" refer to single-stranded RNA (e.g., mature miRNA) or double-stranded RNA (i.e., double-stranded RNA such as siRNA, aiRNA, or pre-miRNA) that, when present in the same cell as the target gene or sequence, can reduce or inhibit the expression of the target gene or sequence (for example, by mediating the degradation of mRNA complementary to the interfering RNA sequence or by inhibiting its translation). Therefore, interfering RNA refers to single-stranded RNA complementary to the target mRNA sequence, or double-stranded RNA formed by two complementary strands or one self-complementary strand. Interfering RNA may have substantial or complete identity with the target gene or sequence, or it may contain mismatched regions (i.e., mismatched motifs). The sequence of the interfering RNA may correspond to the full-length target gene or a subsequence thereof.

[0068] Interfering RNAs include "small interfering RNAs" or "siRNAs," for example, interfering RNAs with a length of approximately 15-60, 15-50, or 15-40 (double-stranded) nucleotides, more typically 15-30, 15-25, or 19-25 (double-stranded) nucleotides, preferably 20-24, 21-22, or 21-23 (double-stranded) nucleotides (e.g., each complementary sequence of a double-stranded siRNA is 1 The siRNA double helix may include a 5-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotide length, preferably about 20-24, 21-22, or 21-23 nucleotide lengths, and the double-stranded siRNA may include a 3' overhang of about 1-4 nucleotides or about 2-3 nucleotides, and a 5' phosphate terminus. Examples of siRNAs include, but are not limited to, double-stranded polynucleotide molecules in which the chain is constructed from two separate molecules, one of which is a sense chain and the other is a complementary antisense chain; double-stranded polynucleotide molecules constructed from single-stranded molecules in which the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; double-stranded polynucleotide molecules having a hairpin secondary structure with self-complementary sense and antisense regions; and cyclic single-stranded polynucleotide molecules having two or more loop structures and a stem having self-complementary sense and antisense regions, wherein the cyclic polynucleotide can be processed in vivo or in vitro to produce an active double-stranded siRNA molecule.

[0069] Preferably, siRNA is chemically synthesized. siRNA can also be produced by cleaving long dsRNA (e.g., dsRNA longer than approximately 25 nucleotides) with E. coli RNase III or Dicer. These enzymes process dsRNA into biologically active siRNAs (see, for example, Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002), Calegari et al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002), Byrom et al., Ambion TechNotes, 10(1):4-6 (2003), Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003), Knight et al., Science, 293:2269-2271 (2001), and Robertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, the dsRNA is at least 50 nucleotides long and about 100, 200, 300, 400, or 500 nucleotides long. The dsRNA may be 1000, 1500, 2000, 5000 nucleotides long or more. The dsRNA can encode an entire gene transcript or a partial gene transcript. In certain cases, siRNA can be encoded by a plasmid (for example, transcribed as a sequence that spontaneously folds into a double helix with a hairpin loop).

[0070] As used herein, the terms “mismatch motif” or “mismatch region” refer to a portion of an interfering RNA (e.g., siRNA, aiRNA, miRNA) sequence that is not 100% complementary to the target sequence. An interfering RNA may have at least one, two, three, four, five, six, or more mismatch regions. Mismatch regions may be contiguous or separated by one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more nucleotides. A mismatch motif or mismatch region may contain one nucleotide or two, three, four, five, or more nucleotides.

[0071] An "effective dose" or "therapeutic effective dose" of nucleic acids (e.g., interfering RNA or mRNA) is an amount sufficient to produce the desired effect, such as inhibition of the expression of a target sequence compared to the normal expression level detected in the absence of interfering RNA, or mRNA-specific expression of a protein that is expressed in vivo and produces the desired biological effect. Inhibition of the expression of a target gene or target sequence is achieved when the value obtained using interfering RNA is approximately 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% of the control. In other embodiments, the expressed protein is the active form of the protein normally expressed in a given cell type in the body, and the therapeutically effective amount of mRNA is the amount that produces a certain amount of the encoded protein, which is at least 50% (e.g., at least 60%, or at least 70%, or at least 80%, or at least 90%) of the amount of protein normally expressed in that cell type in a healthy individual. Suitable assays for measuring the expression of a target gene or target sequence include, for example, testing of protein or RNA levels using techniques known to those skilled in the art, such as dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function testing, and phenotypic assays known to those skilled in the art.

[0072] To “reduce,” “decrease,” “mitigate,” or “reduce” the immune response to interfering RNA is intended to mean a detectable reduction in the immune response to a given interfering RNA (e.g., modified interfering RNA). The amount of reduction in the immune response due to modified interfering RNA can be determined relative to the level of the immune response in the presence of unmodified interfering RNA. A detectable reduction may be approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more lower than the immune response detected in the presence of unmodified interfering RNA. The reduction in the immune response to interfering RNA is typically measured by a decrease in cytokine production by responder cells in vitro (e.g., IFNγ, IFNα, TNFα, IL-6, or IL-12), or by a decrease in cytokine production in the serum of a mammalian subject after administration of interfering RNA.

[0073] To “reduce,” “decrease,” “mitigate,” or “reduce” an mRNA-mediated immune response is intended to mean a detectable reduction in the immune response to a given mRNA (e.g., modified mRNA). The degree of reduction in the immune response due to modified mRNA can be determined relative to the level of the immune response in the presence of unmodified mRNA. A detectable reduction may be approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more lower than the immune response detected in the presence of unmodified mRNA. The reduction in the immune response to mRNA is typically measured by a decrease in cytokine production by responder cells in vitro (e.g., IFNγ, IFNα, TNFα, IL-6, or IL-12) or by a decrease in cytokine production in the serum of a mammalian subject after mRNA administration.

[0074] As used herein, the term "responder cell" refers to a cell, preferably a mammalian cell, that gives rise to a detectable immune response when contacted with an immunostimulatory interfering RNA such as unmodified siRNA. Exemplary responder cells include, for example, dendritic cells, macrophages, peripheral blood mononuclear cells (PBMCs), splenocytes, and the like. Detectable immune responses include, for example, the production of cytokines or growth factors such as TNF-α, IFN-α, IFN-β, IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TGF, and combinations thereof.

[0075] "Substantial identity" refers to a sequence that hybridizes to a reference sequence under stringent conditions or a sequence having a specified percent identity over a particular region of the reference sequence.

[0076] The phrase "stringent hybridization conditions" refers to conditions under which a nucleic acid will typically hybridize to its target sequence but not to other sequences in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. General guidelines for nucleic acid hybridization are found in Tijssen, Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10 °C lower than the thermal melting point (T m ) of a particular sequence at a defined ionic strength and pH. T mThis is the temperature at which 50% of the target-complementary probe hybridizes to the target sequence in equilibrium (at defined ionic strength, pH, and nucleic concentration) (if the target sequence is present in excess, T m (In this case, 50% of the probe is occupied in equilibrium.) Stringent conditions can also be achieved by adding an destabilizer such as formamide. For selective or specific hybridization, the positive signal is at least twice the background, preferably 10 times the background hybridization.

[0077] Exemplary stringent hybridization conditions may include: incubation at 42°C with 50% formamide, 5×SSC, and 1% SDS, or incubation at 65°C with 5×SSC, 1% SDS, followed by washing at 65°C with 0.2×SSC and 0.1% SDS. For PCR, a temperature of approximately 36°C is typical for low-stringency amplification, but the annealing temperature can vary from approximately 32°C to 48°C depending on the primer length. For high-stringency PCR amplification, a temperature of approximately 62°C is typical, but the high-stringency annealing temperature can range from approximately 50°C to approximately 65°C depending on the primer length and specificity. Typical cycle conditions for both high-stringency and low-stringency amplification include a denaturation phase of 30 seconds to 2 minutes at 90°C to 95°C, an annealing phase lasting 30 seconds to 2 minutes, and an extension phase of 1 to 2 minutes at approximately 72°C. Protocols and guidelines for low-stringency and high-stringency amplification reactions are described, for example, in Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. NY (1990).

[0078] Nucleic acids that do not hybridize to one another under stringent conditions remain substantially identical if the polypeptides they encode are substantially identical. This occurs, for example, when copies of nucleic acids are produced using the maximum codon degeneracy permitted by the genetic code. In such cases, nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include hybridization at 37°C in a buffer of 40% formamide, 1M NaCl, and 1% SDS, and washing at 45°C in 1×SSC. Positive hybridization is at least twice the background. Those skilled in the art will readily recognize that similar stringency conditions can be obtained by utilizing alternative hybridization and washing conditions. Further guidelines for determining hybridization parameters are described in numerous references, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds.

[0079] The terms “substantially identical” or “substantially identical” in relation to two or more nucleic acids refer to two or more sequences or subsequences that are the same or have the same nucleotides at a certain percentage (i.e., at least about 60%, preferably at least about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity) when compared and aligned for maximum similarity across a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of sequences when indicated in context. Preferably, substantial identity exists across regions that are at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides long.

[0080] In sequence comparison, typically one sequence acts as a reference sequence, against which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into the computer, and subcoordinates are specified as needed to specify the parameters of the sequence algorithm program. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence to the reference sequence based on the program parameters.

[0081] As used herein, a “comparison window” includes a reference to one of several segments of consecutive positions selected from a group consisting of about 5 to about 60, generally about 10 to about 45, and more generally about 15 to about 30, which allows a given sequence to be compared with a reference sequence of the same number of consecutive positions after the two sequences have been optimally aligned. Methods for sequence alignment for comparison are well known in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970), the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds. (1995 supplement)).

[0082] Preferred examples of algorithms suitable for determining sequence identity percentage and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., Nuc. Acids Res., 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol., 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used to determine the sequence identity percentage of nucleic acids using the parameters described herein. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0083] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indicator of the probability that the match between two nucleotide sequences occurs by chance. For example, if the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0084] As used herein, the term “nucleic acid” refers to polymers containing at least two deoxyribonucleotides or ribonucleotides, in either single-stranded or double-stranded form, and includes DNA and RNA. DNA may be, for example, antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations thereof. RNA may be in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), auto-amplified RNA, and combinations thereof. Nucleic acids include those containing residues or bindings of known nucleotide analogs or modified skeletons, which are synthetic, naturally occurring, and non-natural, and which have similar binding properties to reference nucleic acids. Examples of such analogues include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of native nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes, in addition to the explicitly stated sequence, its conservedly modified variants (e.g., degenerate codon substitutions), alleles, orthologues, SNPs, and complementary sequences. Specifically, degenerate codon substitution can be achieved by constructing sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res., 19:5081 (1991), Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985), Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked to each other via phosphate groups."Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that introduce novel reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides.

[0085] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence containing a partial or full-length coding sequence necessary for the production of a polypeptide or precursor polypeptide.

[0086] As used herein, “gene product” refers to the product of a gene, such as an RNA transcript or polypeptide.

[0087] The term "lipids" refers to a group of organic compounds that include, but are not limited to, fatty acid esters, and are characterized by being insoluble in water but soluble in many organic solvents. They are usually classified into at least three classes: (1) "simple lipids" which include oils and waxes, (2) "complex lipids" which include phospholipids and glycolipids, and (3) "derived lipids" such as steroids.

[0088] The term "alkyl" means, unless otherwise specified, a linear or branched hydrocarbon radical, either alone or as part of another substituent, having the number of carbon atoms indicated (i.e., C1-8 means 1 to 8 carbon atoms), and may be saturated or unsaturated. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. The term "alkenyl" refers to an unsaturated alkyl radical having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated alkyl radical having one or more triple bonds. Examples of unsaturated alkyl groups include vinyl, 2-propenyl, clotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers.

[0089] As used herein, the term lipid nanoparticle "LNP" refers to lipid-nucleic acid particles or nucleic acid-lipid particles (e.g., stable nucleic acid-lipid particles). LNPs are particles made from lipids (e.g., cationic lipids, non-cationic lipids, and complex lipids that prevent particle aggregation) and nucleic acids, wherein nucleic acids (e.g., siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, mRNA, self-amplifying RNA, or plasmids (including plasmids from which interfering RNA or mRNA is transcribed)) are encapsulated within the lipids. In one embodiment, the nucleic acid is encapsulated at least 50% within the lipids; in one embodiment, the nucleic acid is encapsulated at least 75% within the lipids; in one embodiment, the nucleic acid is encapsulated at least 90% within the lipids; and in one embodiment, the nucleic acid is encapsulated completely within the lipids. LNPs typically contain cationic lipids, non-cationic lipids, and lipid conjugates (e.g., PEG-lipid conjugates). LNPs are extremely useful for systemic application because they can exhibit a long circulating life after intravenous (iv) injection, can accumulate at distal sites (e.g., sites physically separated from the injection site), and can mediate the silencing of transfected gene expression or target gene expression at these distal sites.

[0090] The lipid particles (e.g., LNPs) of the present invention typically have an average diameter of about 40 nm to about 150 nm, about 40 nm to about 80 nm, about 40 nm to about 60 nm, about 50 nm to about 60 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm, and are substantially non-toxic. Furthermore, when nucleic acids are present in the lipid particles of the present invention, they are resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for preparing them are disclosed, for example, in U.S. Patent Publications 20040142025 and 20070042031, which are incorporated herein by reference in their entirety for all purposes.

[0091] As used herein, “lipid-encapsulated” may refer to a lipid particle that provides nucleic acids (e.g., interfering RNA or mRNA) that are fully encapsulated, partially encapsulated, or both. In one embodiment, the nucleic acid is fully encapsulated in the lipid particle (e.g., to form an LNP or other nucleic acid-lipid particle).

[0092] The term “cationic lipid” refers to any of a number of lipid species that have a net positive charge at a select pH, such as physiological pH (e.g., pH approximately 7.0). Surprisingly, cationic lipids containing alkyl chains with multiple unsaturated sites, e.g., at least two or three unsaturated sites, have been found to be particularly useful in forming lipid particles with high membrane fluidity. Numerous cationic lipids and related analogues useful in the present invention are described in U.S. Patent Publications 20060083780 and 20060240554; U.S. Patents 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication WO96 / 10390, the disclosures of which are incorporated herein by reference in their entirety for all purposes. Non-limiting examples of cationic lipids are described in detail herein. In some cases, cationic lipids include a protonable tertiary amine (e.g., pH titrable) head group, a C18 alkyl chain, an ether bond between the head group and the alkyl chain, and 0-3 double bonds. Such lipids include, for example, DSDMA, DLinDMA, DLenDMA, and DODMA.

[0093] In the lipid nanoparticles described herein, cationic lipids are, for example, the following: 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), and 2,2-dilinoleyl-4 -(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy- 3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleyl-3-trimethylaminopropane Tylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-stearyloxy-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-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneaminium trifluoroacetate It may be one or more of (DOSPA), dioctadecylamideglycylspermine (DOGS), 3-dimethylamino-2-(cholesta-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadieneoxy)propane (CLinDMA), 2-[5'-(cholesta-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadieneoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), or a mixture thereof. In certain embodiments, the cationic lipid is DLinDMA, DLin-K-C2-DMA ("XTC2"), or a mixture thereof.

[0094] In certain embodiments, the cationic lipids are the following lipids: 3-(((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl)oxy)-N,N-dimethylpropane-1-amine; (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate; (6Z,16Z)-12-((Z)-deca-4-en-1-yl)docosa-6,16dien-11-yl 5-(dimethyl Amino)pentanoate; (6Z,16Z)-12-((Z)-deca-4-en-1-yl)docosa-6,16-dien-11-yl6-(dimethylamino)hexanoate; N,N-dimethyl-4-(tris(((Z)-deca-4-en-1-yl)oxy)silyl)butan-1-amine; N,N-dimethyl-5-(tris(((Z)-deca-4-en-1-yl)oxy)silyl)pentan-1-amine; N,N-dimethyl-6-(tris(((Z)-deca-4-en-1-yl)oxy (C)silyl)hexane-1-amine; 2-(methyl(4-(tris(((Z)-deca-4-en-1-yl)oxy)silyl)butyl)amino)ethane-1-ol; (6Z,16Z)-12-((6-(dimethylamino)hexanoyl)oxy)docosa-6,16-dien-11-yl(Z)-undeca-5-enoate; N1,N3-bis(4-(bis(((Z)-deca-4-en-1-yl)oxy)(methyl)silyl)butyl)-N1,N3-dimethylpropane-1,3-diamine ;N1,N3-dimethyl-N1,N3-bis(4-(tris(((Z)-hepta-3-en-1-yl)oxy)silyl)butyl)propane-1,3-diamine;(1r,4r)-N1,N4-bis(4-(bis(((Z)-deca-4-en-1-yl)oxy)(methyl)silyl)butyl)-N1,N4-dimethylcyclohexane-1,4-diamine;2,8-bis(4-(bis(((Z)-deca-4-en-1-yl)oxy)(methyl)silyl)butyl)-2,8-diazaspiro[4.5] Decane; or bis(2-butyloctyl)10-(N-(3-(dimethylamino)propyl)nonanamide)-nonadecanedioate; di(tridecane-7-yl)10-(N-(3-(dimethylamino)propyl)octanamide)-nonadecanedioate; di(tridecane-7-yl)10-(N-decyl-4-(dimethylamino)butanamide)nonadecanedioate; ((4-hydroxybutyl)azandiyl)bis(nonane-9,1 -Diyl)bis(2-butyloctanoate) or heptadecan-9-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate; 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate or 3,6-bis(4-(bis(2-hydro (Hydroxydodecyl)amino)butyl)piperazine-2,5-dione; 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1-yl)amino)butyl)piperazine-2,5-dione; 1,1'-((2-(1-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperidine-4-yl)ethyl)azandiyl)bis(dodecane-2-ol); te Selected from one of the following: tratridecyl 3,3',3'',3'''-((azandiylbis(propane-3,1-diyl))bis(azantriyl))tetrapropionate, nonyl 8-((8,8-bis(octyloxy)octyl)(2-hydroxyethyl)amino)octanoate, or di((Z)-nona-2-en-1-yl)8,8'-((((2-(dimethylamino)ethyl)thio)carbonyl)azandiyl)dioctanoate.

[0095] The synthesis of cationic lipids such as DLin-K-C2-DMA ("XTC2"), DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, and DLin-K-MPZ, as well as further cationic lipids, is described in U.S. Provisional Application No. 61 / 104,212, filed on 9 October 2008, the disclosure thereof is incorporated herein by reference in its entirety for all purposes. The synthesis of cationic lipids such as DLin-K-DMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl, DLin-MPZ, DLinAP, DOAP, and DLin-EG-DMA, as well as further cationic lipids, is described in PCT application PCT / US08 / 88676, filed on 31 December 2008, and its disclosure is incorporated herein by reference in its entirety for all purposes. The synthesis of cationic lipids such as CLinDMA, as well as further cationic lipids, is described in U.S. Patent Publication 20060240554, and its disclosure is incorporated herein by reference in its entirety for all purposes.

[0096] Any various cationic lipids can be used alone or in combination with one or more other cationic or non-cationic lipid species in the lipid particles (e.g., LNPs) of the present invention.

[0097] The cationic lipids useful in the present invention may be any of a number of lipid species having a net positive charge at physiological pH. Such lipids include 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- (1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyroxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-1-propane Aminonium trifluoroacetate (DOSPA), dioctadecylamide glycylspermine (DOGS), 3-dimethylamino-2-(cholesta-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecanedienoxy)propane (CLinDMA), 2-[5'-(cholesta-5-ene-3.beta)-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecanedienoxy) Examples include, but are not limited to, xy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamyl-3-dimethylaminopropane (DLinCDAP), and mixtures thereof.Numerous of these lipids and related analogues are described in U.S. Patent Publications 20060083780 and 20060240554; U.S. Patents 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613, and 5,785,992; and PCT Publication WO96 / 10390, the disclosures of which are incorporated herein by reference in their entirety for any purpose. Furthermore, numerous commercially available preparations of cationic lipids are available and can be used in the present invention. These include, for example, LIPOFECTIN® (a commercially available cationic liposome containing DOTMA and DOPE, GIBCO / BRL, Grand Island, NY, USA); LIPOFECTAMINE® (a commercially available cationic liposome containing DOSPA and DOPE, GIBCO / BRL); and TRANSFECTAM® (a commercially available cationic liposome containing DOGS, Promega Corp., Madison, Wis., USA).

[0098] Furthermore, cationic lipids of formula I having the following structure are useful in the present invention. [ka] Here, R 1 and R 2 is independently selected and is H or C1-C3 alkyl, and R 3 and R 4 R is independently selected and is an alkyl group having approximately 10 to approximately 20 carbon atoms. 3 and R 4 At least one of them contains at least two unsaturated sites. In certain cases, R 3 and R 4 Both are the same, that is, R 3 and R 4 Both are Rinorail (C 18 ) etc. In other specific cases, R 3 and R 4 They are different, that is, R 3 is tetradectrienyl (C14 ) and R 4 is Rinorail (C 18 ) In one embodiment, the cationic lipid of formula I is symmetric, that is, R 3 and R 4 Both are the same. In another embodiment, R 3 and R 4 Both contain at least two unsaturated sites. In some embodiments, R 3 and R 4 is independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadieniyl. In one embodiment, R 3 and R 4 Both are linoleic. In some embodiments, R 3 and R 4 It comprises at least three unsaturated moieties, independently selected from, for example, dodecatrienyl, tetradectorienyl, hexadecatrienyl, linolenyl, and eicosatrienyl. In certain embodiments, the cationic lipid of formula I is 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) or 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0099] Furthermore, cationic lipids of formula II having the following structure are useful in the present invention. [ka] Here, R 1 and R 2 is independently selected and is H or C1-C3 alkyl, and R 3 and R 4 R is independently selected and is an alkyl group having approximately 10 to approximately 20 carbon atoms. 3 and R 4 At least one of them contains at least two unsaturated sites. In certain cases, R 3 and R 4 Both are the same, that is, R 3 and R 4 Both are Rinorail (C18 ) etc. In other specific cases, R 3 and R 4 They are different, that is, R 3 is tetradectrienyl (C 14 ) and R 4 is Rinorail (C 18 ) In one embodiment, the cationic lipid of the present invention is symmetric, i.e., R 3 and R 4 Both are the same. In another embodiment, R 3 and R 4 Both contain at least two unsaturated sites. In some embodiments, R 3 and R 4 is independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadieniyl. In one embodiment, R 3 and R 4 Both are linoleic. In some embodiments, R 3 and R 4 It comprises at least three unsaturated sites, independently selected from, for example, dodecatrienyl, tetradectorienyl, hexadecatrienyl, linolenyl, and icosatrienyl.

[0100] Furthermore, cationic lipids of formula III having the following structure (or their salts) are useful in the present invention. [ka] Here, R 1 and R 2 C may be the same or different, independently, and may be substituted. 12 -C 24 Alkyl, possibly substituted C 12 -C 24 Alkenyl, C may be substituted 12 -C 24 Alkynyl or possibly substituted C 12 -C 24 It is Asil; R 3 and R 4These are the same or different, independently, a substituted C1-C6 alkyl, a substituted C1-C6 alkenyl, or a substituted C1-C6 alkynyl, or R 3 and R 4 They may bond to form a heterocycle which may be substituted with 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen; R 5 is either absent or hydrogen or a C1-C6 alkyl group, providing a quaternary amine; m, n, and p are the same or different, independently either 0 or 1, provided that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; Y and Z are the same or different, independently either O, S, or NH.

[0101] In some embodiments, the cationic lipid of formula III is 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminoethyl Tylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin- DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleyl These are 3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), or mixtures thereof. In some embodiments, the cationic lipid of formula III is DLin-K-C2-DMA(XTC2).

[0102] Cationic lipids may constitute approximately 50 mol% to 90 mol%, 50 mol% to 85 mol%, 50 mol% to 80 mol%, 50 mol% to 75 mol%, 50 mol% to 70 mol%, 50 mol% to 65 mol%, or 55 mol% to 65 mol% of the total lipids present in the particles.

[0103] It will be readily apparent to those skilled in the art that the proportion of components can be varied depending on the intended use of the particles, and that the delivery efficiency of a particular formulation can be measured, for example, using an endosomal release parameter (ERP) assay.

[0104] The noncationic lipids used in the lipid particles (e.g., LNPs) of the present invention can be any variety of neutral, uncharged, zwitterionic, or anionic lipids capable of forming stable complexes.

[0105] Non-cationic lipids include, but are not limited to, lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine Phospholipids include nolamines (POPE), palmitoyl oleioyl phosphatidylglycerol (POPG), dioleoyl phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), monomethyl phosphatidylethanolamine, dimethyl phosphatidylethanolamine, dierydoyl phosphatidylethanolamine (DEPE), stearoyl oleioyl phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl group in these lipids is typically C 10 -C 24 The acyl group is derived from a fatty acid having a carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0106] Further examples of noncationic lipids include sterols such as cholesterol, and their derivatives, such as cholestanol, cholestanone, cholestane, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

[0107] In some embodiments, the noncationic lipids present in the lipid particles (e.g., LNPs) include or consist of cholesterol or its derivatives, for example, phospholipid-free lipid particle formulations. In other embodiments, the noncationic lipids present in the lipid particles (e.g., LNPs) include or consist of one or more phospholipids, for example, cholesterol-free lipid particle formulations. In further embodiments, the noncationic lipids present in the lipid particles (e.g., LNPs) include or consist of a mixture of one or more phospholipids and cholesterol or its derivatives.

[0108] Other examples of noncationic lipids suitable for use in the present invention include phosphorus-free lipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, and sphingomyelin.

[0109] The term "hydrophobic lipid" refers to compounds having nonpolar groups, including but not limited to saturated and unsaturated long-chain aliphatic hydrocarbon groups, and those in which such groups are optionally substituted with one or more aromatic, alicyclic, or heterocyclic groups. Preferred examples include, but are not limited to, diacylglycerol, dialkylglycerol, NN-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0110] The term "membrane fusion" refers to the ability of lipid particles, such as LNPs, to fuse with the cell membrane. The membrane can be either the plasma membrane or the membrane surrounding organelles, such as endosomes or the nucleus.

[0111] As used herein, the term "aqueous solution" refers to a composition consisting of water in whole or in part.

[0112] As used herein, the term “organolipid solution” refers to a composition comprising an organic solvent that is entirely or partially lipid-containing.

[0113] As used herein, "distal region" refers to a physically separated region and is not limited to adjacent capillary beds, but includes regions widely distributed throughout the body.

[0114] "Serum stability" in relation to lipid nanoparticles such as LNPs means that the particles are not significantly degraded after exposure to serum assays or nuclease assays that significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0115] As used herein, “systemic delivery” refers to the delivery of lipid particles that result in widespread biodistribution of nucleic acids, such as interfering RNA or mRNA, within the body. Some administration techniques can result in systemic delivery of a particular drug, while others cannot. Systemic delivery means that a useful amount, preferably a therapeutic amount, of the drug is exposed to a large portion of the body. To achieve widespread biodistribution, a blood lifetime is generally required so that the drug does not undergo rapid degradation or clearance (by first-pass organs (such as the liver or lungs) or rapid nonspecific cell binding, etc.) before reaching disease sites distal to the administration site. Systemic delivery of lipid particles may be by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal delivery. In preferred embodiments, systemic delivery of lipid particles is by intravenous delivery.

[0116] As used herein, "local delivery" refers to the direct delivery of nucleic acids, such as interfering RNA or mRNA, to a target site within a living organism. For example, a drug can be locally delivered by direct injection to a disease site such as a tumor, or to another target site such as an inflammatory site, or to a target organ such as the liver, heart, pancreas, or kidney.

[0117] The term "mammal" refers to any species of mammal, such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, and domesticated animals.

[0118] The term "cancer" refers to any member of the class of diseases characterized by the uncontrolled proliferation of abnormal cells. This term includes all known cancers and neoplastic diseases, regardless of whether their features are malignant, benign, soft tissue, or solid, and cancers at all stages and grades, including pre-metastatic and post-metastatic. Examples of different types of cancer include, but are not limited to, lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, stomach (gastric) cancer, esophageal cancer; gallbladder cancer, liver cancer, pancreatic cancer, appendiceal cancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer, kidney cancer (e.g., renal cell carcinoma), central nervous system cancers, glioblastoma, skin cancer, lymphoma, choriocarcinoma, head and neck cancer, osteogenic sarcoma, and hematological cancers. Non-limiting examples of specific types of liver cancer include hepatocellular carcinoma (HCC), secondary liver cancer (e.g., resulting from metastasis of several other non-hepatocellular carcinoma cell types), and hepatoblastoma. As used herein, “tumor” includes one or more cancerous cells.

[0119] Certain embodiments provide a pharmaceutical composition comprising lipid nanoparticles described herein and a pharmaceutically acceptable carrier.

[0120] In certain embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0121] Certain embodiments provide a method for delivering nucleic acids to cells, comprising contacting the cells with lipid nanoparticles described herein.

[0122] A particular embodiment provides a method for treating a disease characterized by a deficiency of a functional protein (e.g., caused by a genetic defect), comprising: administering lipid nanoparticles described herein to a subject having the disease, wherein the lipid nanoparticles comprise a functional protein or mRNA encoding a protein having the same biological activity as the functional protein.

[0123] A particular embodiment provides a method for treating a disease characterized by the overexpression of a polypeptide, comprising administering lipid nanoparticles described herein to a subject having the disease, wherein the lipid nanoparticles include an siRNA that targets the expression of the overexpressed polypeptide.

[0124] Certain embodiments provide lipid nanoparticles described herein for therapeutic or prophylactic treatment of diseases characterized by deficiencies in functional proteins (e.g., caused by genetic defects).

[0125] Certain embodiments provide lipid nanoparticles described herein for therapeutic or prophylactic treatment of diseases characterized by polypeptide overexpression.

[0126] In certain embodiments, nucleic acids are completely encapsulated within the lipid portion of the lipid particles so that the nucleic acids in the lipid particles are resistant to enzymatic degradation by, for example, nucleases or proteases in aqueous solution. In certain other embodiments, the lipid particles are substantially non-toxic to mammals such as humans.

[0127] In certain cases, nucleic acids include interfering RNA molecules such as siRNA, aiRNA, miRNA, or mixtures thereof. In certain other cases, nucleic acids include single-stranded or double-stranded DNA, RNA, or DNA / RNA hybrids such as antisense oligonucleotides, ribozymes, plasmids, immunostimulatory oligonucleotides, or mixtures thereof. In certain other cases, nucleic acids include one or more mRNA molecules (e.g., cocktails).

[0128] In one embodiment, the nucleic acid comprises siRNA. In one embodiment, the siRNA molecule comprises a double-stranded region of about 15 to about 60 nucleotides in length (e.g., about 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 25, or 19 to 25 nucleotides, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides). The siRNA molecule of the present invention has the ability to silence the expression of a target sequence in vitro and / or in vivo.

[0129] In some embodiments, the siRNA molecule contains at least one modified nucleotide. In certain preferred embodiments, the siRNA molecule contains one, two, three, four, five, six, seven, eight, nine, ten, or more modified nucleotides in the double-stranded region. In certain cases, the siRNA contains about 1% to about 100% (e.g., about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in the double-stranded region. In a preferred embodiment, less than 25% (e.g., less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%) of the nucleotides in the double-stranded region include modified nucleotides.

[0130] In other embodiments, the siRNA molecule includes, but is 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. In preferred embodiments, the siRNA includes 2'OMe nucleotides (e.g., 2'OMe purine and / or pyrimidine nucleotides), such as 2'OMe-guanosine nucleotide, 2'OMe-uridine nucleotide, 2'OMe-adenosine nucleotide, and 2'OMe-cytosine nucleotide, as well as mixtures thereof. In certain cases, the siRNA does not include 2'OMe-cytosine nucleotide. In other embodiments, the siRNA includes a hairpin loop structure.

[0131] siRNA may contain modified nucleotides on one strand (i.e., sense or antisense) or both strands of the double-stranded region of the siRNA molecule. Preferably, uridine nucleotides and / or guanosine nucleotides are modified at selective positions in the double-stranded region of the siRNA double helix. With respect to uridine nucleotide modification, at least 1, 2, 3, 4, 5, 6 or more uridine nucleotides on the sense strand and / or antisense strand may be modified uridine nucleotides such as 2'OMe-uridine nucleotides. In some embodiments, all uridine nucleotides on the sense strand and / or antisense strand are 2'OMe-uridine nucleotides. With respect to guanosine nucleotide modification, at least 1, 2, 3, 4, 5, 6 or more guanosine nucleotides on the sense strand and / or antisense strand may be modified guanosine nucleotides such as 2'OMe-guanosine nucleotides. In some embodiments, all guanosine nucleotides on the sense strand and / or antisense strand are 2'OMe-guanosine nucleotides.

[0132] In certain embodiments, at least one, two, three, four, five, six, seven, or more 5'-GU-3' motifs in the siRNA sequence may be modified, for example, by introducing mismatches to remove 5'-GU-3' motifs and / or by introducing modified nucleotides such as 2'OMe nucleotides. The 5'-GU-3' motifs may be present in the sense strand, the antisense strand, or both strands of the siRNA sequence. The 5'-GU-3' motifs may be adjacent to each other, or instead separated by one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more nucleotides.

[0133] In some preferred embodiments, modified siRNA molecules are less immunostimulant than their corresponding unmodified siRNA sequences. In such embodiments, the modified siRNA molecules with reduced immunostimulant properties conveniently retain RNAi activity against the target sequence. In other embodiments, the immunostimulant properties of a modified siRNA molecule and its ability to silence target gene expression can be balanced or optimized by introducing minimal and selective 2'OMe modifications within the siRNA sequence, such as the double-stranded region of an siRNA double helix. In specific cases, the modified siRNA is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% less immunostimulant than the corresponding unmodified siRNA. It will be readily apparent to those skilled in the art that the immunostimulatory properties of modified siRNA molecules and their corresponding unmodified siRNA molecules can be determined, for example, by measuring INF-α and / or IL-6 levels approximately 2 to 12 hours after systemic administration in mammals or transfection of mammalian responder cells using a suitable lipid-based delivery system (such as the LNP delivery system disclosed herein).

[0134] In certain embodiments, the modified siRNA molecule is IC50 (i.e., the median inhibitory concentration) is less than one-tenth that of the corresponding unmodified siRNA (i.e., the modified siRNA is IC 50 However, the IC of the corresponding unmodified siRNA 50 (It is less than one-tenth of that). In other embodiments, the modified siRNA is IC 50 However, this is less than one-third of that of the corresponding unmodified siRNA sequence. In yet another embodiment, the modified siRNA is IC 50 However, it is less than half that of the corresponding unmodified siRNA. Dose-response curves can be generated using methods known to those skilled in the art, as well as ICs for modified siRNA and the corresponding unmodified siRNA. 50 It will be readily apparent to those skilled in the art that the value can be easily determined.

[0135] In yet another embodiment, the modified siRNA molecule has the ability to silence at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the expression of the target sequence compared to the corresponding unmodified siRNA sequence.

[0136] In some embodiments, the siRNA molecule does not contain phosphate backbone modifications, for example, in the sense and / or antisense strands of the double-stranded region. In other embodiments, the siRNA contains one, two, three, four, or more phosphate backbone modifications, for example, in the sense and / or antisense strands of the double-stranded region. In preferred embodiments, the siRNA does not contain phosphate backbone modifications.

[0137] In further embodiments, the siRNA does not contain 2'-deoxynucleotides in, for example, the sense strand and / or antisense strand of the double-stranded region. In further embodiments, the siRNA contains, for example, one, two, three, four, or more 2'-deoxynucleotides in the sense strand and / or antisense strand of the double-stranded region. In preferred embodiments, the siRNA does not contain 2'-deoxynucleotides.

[0138] In certain cases, the nucleotides at the 3' end of the double-stranded region of the sense strand and / or antisense strand are not modified nucleotides. In other certain cases, the nucleotides near the 3' end of the double-stranded region of the sense strand and / or antisense strand (e.g., within 1, 2, 3, or 4 nucleotides from the 3' end) are not modified nucleotides.

[0139] The siRNA molecules described herein may have 3' overhangs of 1, 2, 3, 4, or more nucleotides on one or both sides of the double-stranded region, or they may lack overhangs on one or both sides of the double-stranded region (i.e., they may have blunt ends). Preferably, the siRNA has 2-nucleotide 3' overhangs on either side of the double-stranded region. In certain cases, the 3' overhang on the antisense strand is complementary to the target sequence, and the 3' overhang on the sense strand is complementary to the complementary strand of the target sequence. Alternatively, the 3' overhang is not complementary to either the target sequence or its complementary strand. In some embodiments, the 3' overhang contains one, two, three, four, or more nucleotides, such as 2'-deoxy(2'H) nucleotides. In certain preferred embodiments, the 3' overhang contains deoxythymidine (dT) and / or uridine nucleotides. In other embodiments, one or more nucleotides in the 3' overhangs on one or both sides of the double-stranded region include modified nucleotides. Non-limiting examples of modified nucleotides have been described above and include 2'OMe nucleotides, 2'-deoxy-2'F nucleotides, 2'-deoxy nucleotides, 2'-O-2-MOE nucleotides, LNA nucleotides, and mixtures thereof. In preferred embodiments, one, two, three, four, or more nucleotides in the 3' overhangs present on the sense and / or antisense strands of the siRNA include, for example, 2'OMe nucleotides (e.g., 2'OMe purines and / or pyrimidine nucleotides) such as 2'OMe-guanosine nucleotide, 2'OMe-uridine nucleotide, 2'OMe-adenosine nucleotide, 2'OMe-cytosine nucleotide, and mixtures thereof.

[0140] siRNA may contain at least one unmodified and / or modified siRNA sequence, or a cocktail thereof (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more), that silence the expression of a target gene. The siRNA cocktail may contain sequences targeting the same region or domain (e.g., a "hotspot") and / or different regions or domains of one or more target genes. In certain cases, one or more modified siRNAs (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) that silence the expression of the target gene are present in the cocktail. In other certain cases, one or more unmodified siRNA sequences (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) that silence the expression of the target gene are present in the cocktail.

[0141] In some embodiments, the antisense strand of the siRNA molecule contains 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 contains 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 contains or consists of a sequence that specifically hybridizes to the target sequence or a portion thereof.

[0142] In further embodiments, the sense strand of the siRNA molecule contains 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 contains or consists of a sequence that is 100% identical to the target sequence or a portion thereof.

[0143] Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof. The synthesis of cholesteryl-2'-hydroxyethyl ether is described herein.

[0144] As used herein, DSPC means distearoylphosphatidylcholine.

[0145] In the lipid particles of the present invention, nucleic acids are completely encapsulated within the lipid portion of the particle, thereby protecting the nucleic acids from enzymatic degradation. In preferred embodiments, LNPs containing nucleic acids such as interfering RNA (e.g., siRNA) or mRNA are completely encapsulated within the lipid portion of the particle, thereby protecting the nucleic acids from nuclease degradation. In certain cases, the nucleic acids in the LNPs remain substantially undegraded after the particles are exposed to nucleases at 37°C for at least about 20, 30, 45, or 60 minutes. In certain other cases, the nucleic acids in the LNPs remain substantially undegraded after the particles are incubated in serum at 37°C for at least about 30, 45, or 60 minutes or for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the nucleic acids (e.g., nucleic acids such as siRNA or mRNA) form a complex with the lipid portion of the particle. One of the advantages of the formulation of the present invention is that the lipid particle composition is substantially non-toxic to mammals such as humans.

[0146] The term "fully encapsulated" indicates that the nucleic acids in the lipid particles are not significantly degraded after exposure to serum or nuclease or protease assays that can significantly degrade free DNA, RNA, or proteins. In a fully encapsulated system, in a process that typically degrades 100% of free nucleic acids, preferably less than about 25% of the nucleic acids in the particles are degraded, more preferably less than about 10%, and most preferably less than about 5%. In relation to nucleic acid therapeutics, fully encapsulated can be determined by the Oligreen® assay. Oligreen® is a highly sensitive fluorescent nucleic acid stain for quantifying oligonucleotides and single-stranded DNA or RNA in solution (available from Invitrogen Corporation; Carlsbad, Calif.). "Fully encapsulated" also indicates that the lipid particles are stable in serum, i.e., they are not rapidly degraded into their components upon in vivo administration.

[0147] In another aspect, the present invention provides a lipid particle (e.g., LNP) composition comprising a plurality of lipid particles. In a preferred embodiment, nucleic acids (e.g., nucleic acids) make up about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 30% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 30% to about 90%, and about 40% to about 90% of the lipid particles (e.g., LNP). %, approximately 50% to 90%, approximately 60% to 90%, approximately 70% to 90%, approximately 80% to 90%, or at least approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or range within that) are completely encapsulated within the lipid portion of the lipid particle (e.g., LNP).

[0148] Typically, the lipid particles (e.g., LNPs) of the present invention have a lipid:activator (e.g., lipid:nucleic acid) ratio (mass / mass ratio) of about 1 to about 100. In some cases, the lipid:activator (e.g., lipid:nucleic acid) ratio (mass / mass ratio) is in the range of about 1 to about 50, about 2 to about 25, about 3 to about 20, about 4 to about 15, or about 5 to about 10.

[0149] Typically, the lipid particles (e.g., LNPs) of the present invention have an average diameter of about 40 nm to about 150 nm. In some embodiments, the lipid particles of the present invention have an average diameter of about 40 nm to about 130 nm, about 40 nm to about 120 nm, about 40 nm to about 100 nm, about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 40 nm to about 80 nm, about 40 nm to about 60 nm, about 50 nm to about 60 nm, about 60 nm to about 120 nm, about 60 nm to about 110 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 70 nm to about 120 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, or about 150 nm, 120 nm, 110 nm, 100 nm, 90 nm, or less than 80 nm (or any fraction thereof or a range within those). The present invention also provides pharmaceutical compositions comprising lipid particles (e.g., LNPs) and pharmaceutically acceptable carriers as described herein.

[0150] In further embodiments, the present invention provides a method for introducing one or more active agents or therapeutic agents (e.g., nucleic acids) into cells, comprising contacting the cells with lipid particles (e.g., LNPs) as described herein. In one embodiment, the cells are in a mammal, and the mammal is a human. In another embodiment, the present invention provides a method for in vivo delivery of one or more active agents or therapeutic agents (e.g., nucleic acids), comprising administering lipid particles (e.g., LNPs) as described herein to a mammalian subject. In preferred embodiments, the mode of administration includes, but is not limited to, oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intrafocal, intratracheal, subcutaneous, and intradermal. Preferably, the mammalian subject is a human.

[0151] In one embodiment, at least about 5%, 10%, 15%, 20%, or 25% of the total injected dose of lipid particles (e.g., LNPs) are present in the plasma about 8, 12, 24, 36, or 48 hours after injection. In other embodiments, more than about 20%, more than 30%, more than 40%, and about 60%, 70%, or 80% of the total injected dose of lipid particles (e.g., LNPs) are present in the plasma about 8, 12, 24, 36, or 48 hours after injection. In certain cases, more than about 10% of the particles are present in the mammalian plasma about 1 hour after administration. In certain other cases, the presence of lipid particles (e.g., LNPs) is detectable at least about 1 hour after administration of the particles. In certain embodiments, the presence of nucleic acids such as interfering RNA (e.g., siRNA) or mRNA can be detected intracellularly approximately 8, 12, 24, 36, 48, 60, 72, or 96 hours after administration (e.g., lung, liver, tumor, or inflammatory site). In other embodiments, downregulation of target sequence expression by nucleic acids such as interfering RNA (e.g., siRNA) can be detected approximately 8, 12, 24, 36, 48, 60, 72, or 96 hours after administration. In yet another embodiment, downregulation of target sequence expression by nucleic acids such as interfering RNA (e.g., siRNA) preferentially occurs in tumor cells or in cells at an inflammatory site. In further embodiments, the presence or action of nucleic acids such as interfering RNA (e.g., siRNA) within cells in a site proximal or distal to the administration site, or within cells of the lung, liver, or tumor, can be detected approximately 12, 24, 48, 72, or 96 hours after administration, or approximately 6, 8, 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days after administration. In other embodiments, upregulation of target sequence expression by nucleic acids such as mRNA or auto-amplified RNA can be detected approximately 8, 12, 24, 36, 48, 60, 72, or 96 hours after administration. In yet another embodiment, upregulation of target sequence expression by nucleic acids such as mRNA or auto-amplified RNA preferentially occurs within tumor cells or within cells at the site of inflammation.In further embodiments, the presence or action of nucleic acids such as mRNA or auto-amplified RNA in cells of a site proximal or distal to the administration site, or in cells of the lung, liver, or tumor, can be detected approximately 12, 24, 48, 72, or 96 hours after administration, or approximately 6, 8, 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days after administration. In further embodiments, the lipid particles of the present invention (e.g., LNPs) are administered parenterally or intraperitoneally. In embodiments, the lipid particles of the present invention (e.g., LNPs) are administered intramuscularly.

[0152] In some embodiments, the lipid particles (e.g., LNPs) of the present invention are useful for methods of therapeutic delivery of one or more nucleic acids, including interfering RNA sequences (e.g., siRNA). In particular, one object of the present invention is to provide in vitro and in vivo methods for treating diseases or disorders in mammals (e.g., rodents such as mice, or primates such as humans, chimpanzees, or monkeys) by downregulating or silencing the transcription and / or translation of one or more target nucleic acid sequences or target genes. As a non-limiting example, the methods of the present invention are useful for in vivo delivery of interfering RNA (e.g., siRNA) to the liver and / or tumors of mammalian subjects. In certain embodiments, the disease or disorder is associated with gene expression and / or overexpression, and gene expression or overexpression is reduced by interfering RNA (e.g., siRNA). In other specific embodiments, a therapeutically effective amount of lipid particles (e.g., LNPs) may be administered to a mammal. In some cases, interfering RNA (e.g., siRNA) is formulated within LNPs, and these particles are administered to patients requiring such treatment. In other cases, cells are removed from the patient, interfering RNA (e.g., siRNA) is delivered in vitro (e.g., using LNPs as described herein), and the cells are reinjected into the patient.

[0153] In additional embodiments, the present invention provides lipid particles (e.g., LNPs) containing asymmetric interfering RNA (aiRNA) molecules for silencing the expression of a target gene, and a method for silencing the expression of a target gene using such particles.

[0154] In one embodiment, the aiRNA molecule comprises a double-stranded region approximately 10 to 25 base pairs long, and the aiRNA molecule includes an antisense strand with 5' and 3' overhangs, and the aiRNA molecule has the ability to silence target gene expression.

[0155] In certain cases, the aiRNA molecule contains a double-stranded region with a nucleotide length of approximately 12–20, 12–19, 12–18, 13–17, or 14–17 (base pairs), more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 (base pairs) nucleotides. In other specific cases, the 5' and 3' overhangs on the antisense strand contain a sequence complementary to the target RNA sequence and may optionally further contain a non-target sequence. In some embodiments, each of the 5' and 3' overhangs on the antisense strand contains or consists of one, two, three, four, five, six, seven, or more nucleotides.

[0156] In other embodiments, the aiRNA molecule comprises a modified nucleotide selected from the group consisting of 2'OMe nucleotide, 2'F nucleotide, 2'-deoxynucleotide, 2'-O-MOE nucleotide, LNA nucleotide, and mixtures thereof. In preferred embodiments, the aiRNA molecule comprises a 2'OMe nucleotide. As a non-limiting example, the 2'OMe nucleotide may be selected from the group consisting of 2'OMe-guanosine nucleotide, 2'OMe-uridine nucleotide, and mixtures thereof.

[0157] In related embodiments, the present invention provides lipid particles (e.g., LNPs) containing microRNA (miRNA) molecules that silence the expression of a target gene, and a method for silencing the expression of a target gene using such a composition.

[0158] In one embodiment, the miRNA molecule has a length of approximately 15 to 60 nucleotides and has the ability to silence the expression of a target gene.

[0159] In certain cases, the miRNA molecule has a length of about 15–50, 15–40, or 15–30 nucleotides, more typically about 15–25 or 19–25 nucleotides, preferably about 20–24, 21–22, or 21–23 nucleotides. In preferred embodiments, the miRNA molecule is a mature miRNA molecule that targets a target RNA sequence.

[0160] In some embodiments, the miRNA molecule comprises a modified nucleotide selected from the group consisting of 2'OMe nucleotide, 2'F nucleotide, 2'-deoxynucleotide, 2'-O-MOE nucleotide, LNA nucleotide, and mixtures thereof. In preferred embodiments, the miRNA molecule comprises a 2'OMe nucleotide. In non-limiting examples, the 2'OMe nucleotide may be selected from the group consisting of 2'OMe-guanosine nucleotide, 2'OMe-uridine nucleotide, and mixtures thereof.

[0161] In some embodiments, the lipid particles (e.g., LNPs) of the present invention are useful in methods for the therapeutic delivery of one or more mRNA molecules. In particular, one object of the present invention is to provide in vitro and in vivo methods for the treatment of diseases or disorders in mammals (e.g., rodents such as mice or primates such as humans, chimpanzees, or monkeys) through the expression of one or more target proteins. As a non-limiting example, the methods of the present invention are useful for the in vivo delivery of one or more mRNA molecules to mammalian subjects. In other specific embodiments, a therapeutically effective amount of lipid particles (e.g., LNPs) may be administered to a mammal. In some cases, one or more mRNA molecules are formulated in LNPs, and these particles are administered to a patient in need of such treatment. In other cases, cells are removed from the patient, one or more mRNA molecules are delivered in vitro (e.g., using LNPs as described herein), and the cells are reinjected into the patient.

[0162] In other embodiments, the siRNA molecule comprises a modified nucleotide selected from the group consisting of 2'OMe nucleotides, 2'F nucleotides, 2'-deoxynucleotides, 2'-O-MOE nucleotides, LNA nucleotides, and mixtures thereof. In related embodiments, the present invention provides lipid particles (e.g., LNPs) containing microRNA (miRNA) molecules for silencing the expression of a target gene, and a method for silencing the expression of a target gene using such a composition.

[0163] Therefore, the lipid particles (e.g., LNPs) of the present invention are stable in circulation, are of a size required for pharmacodynamic behavior that provides access to the extravascular site, and can reach target cell populations, making them convenient and suitable for use in the administration of activators or therapeutic agents such as nucleic acids (e.g., interfering RNAs such as siRNA, aiRNA, and / or miRNA, or mRNA; guide RNA; self-amplifying RNA; circular RNA; DNA, e.g., plasmid DNA and closed-ended DNA) to subjects (e.g., mammals such as humans)

[0164] In relation to the present invention, the terms "polynucleotide" and "oligonucleotide" refer to polymers or oligomers of nucleotides or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (skeletal) bonds. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers, or portions thereof, that contain similarly functional monomers that do not exist in nature. Such modified or substituted oligonucleotides are often preferred over their natural counterparts due to properties such as improved intracellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0165] Oligonucleotides are generally classified into deoxyribooligonucleotides or ribooligonucleotides. Deoxyribooligonucleotides are composed of a 5-carbon sugar called deoxyribose, which is covalently bonded to a phosphate group at its 5' and 3' carbon atoms, forming an unbranched alternating polymer. Ribooligonucleotides consist of a similar repeating structure in which the 5-carbon sugar is ribose.

[0166] The nucleic acids present in the lipid-nucleic acid particles according to the present invention include any known form of nucleic acid. The nucleic acids used herein may be single-stranded DNA or RNA, double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double-stranded DNA are described herein and include, for example, structural genes, genes containing regulatory and termination regions, and self-replicating systems such as viral DNA or plasmid DNA. Examples of double-stranded RNA are described herein and include, for example, siRNA, and other RNAi agents such as aiRNA and pre-miRNA. Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, mature miRNAs, and triple-helix-forming oligonucleotides.

[0167] Nucleic acids can generally be of varying lengths depending on the specific form of the nucleic acid. For example, in certain embodiments, a plasmid or gene may be about 1,000 to about 100,000 nucleotides long. In certain embodiments, an oligonucleotide may be in the range of about 10 to about 100 nucleotides long. In various related embodiments, oligonucleotides, whether single-stranded, double-stranded, or triple-stranded, may be in the range of about 10 to about 60 nucleotides, about 15 to about 60 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 30 nucleotides long.

[0168] In certain embodiments, the oligonucleotide (or its chain) of the present invention specifically hybridizes to or is complementary to a target polynucleotide sequence. As used herein, the terms “specifically hybridizable” and “complementary” refer to a sufficient degree of complementarity that results in stable and specific binding between the target DNA or RNA and the oligonucleotide. It should be understood that an oligonucleotide does not need to be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. In preferred embodiments, an oligonucleotide is specifically hybridizable if its binding to the target sequence interferes with the normal function of the target sequence, causing a loss of usefulness or expression from the target sequence, and if there is sufficient complementarity to avoid nonspecific binding of the oligonucleotide to a non-target sequence under the conditions under which specific binding is desired—i.e., physiological conditions in the case of an in vivo assay or therapeutic treatment, or under the conditions under which the assay is performed in the case of an in vitro assay. Therefore, an oligonucleotide may contain one, two, three, or more base substitutions compared to the region of the gene or mRNA sequence it targets or with which it specifically hybridizes.

[0169] siRNA The siRNA component of the lipid nanoparticles of the present invention has the ability to silence the expression of a target gene of interest. Each strand of the siRNA duplex is typically about 15 to about 60 nucleotides in length, preferably about 15 to about 30 nucleotides in length. In certain embodiments, the siRNA contains at least one modified nucleotide. Modified siRNAs generally have less immunostimulation than the corresponding unmodified siRNA sequences and retain RNAi activity against the target gene of interest. In some embodiments, the modified siRNA contains at least one 2'-OMe purine nucleotide or 2'-OMe pyrimidine nucleotide, such as 2'-OMe-guanosine nucleotide, 2'-OMe-uridine nucleotide, 2'-OMe-adenosine nucleotide, and / or 2'-OMe-cytosine nucleotide. In a preferred embodiment, one or more of the uridine and / or guanosine nucleotides are modified. The modified nucleotides can be present in one strand (i.e., sense or antisense) or both strands of the siRNA. The siRNA sequence may have an overhang (e.g., a 3' or 5' overhang as described in Elbashir et al., Genes Dev., 15:188 (2001) or Nykanen et al., Cell, 107:309 (2001)) or may lack an overhang (i.e., have blunt ends).

[0170] Modified siRNAs generally contain from about 1% to about 100% (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in the double-stranded region of the siRNA duplex. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the nucleotides within the double-stranded region of the siRNA contain modified nucleotides.

[0171] In some embodiments, less than approximately 25% of the nucleotides in the double-stranded region of the siRNA (e.g., less than approximately 25%, less than approximately 24%, less than approximately 23%, less than approximately 22%, less than approximately 21%, less than approximately 20%, less than approximately 19%, less than approximately 18%, less than approximately 17%, less than approximately 16%, less than approximately 15%, less than approximately 14%, less than approximately 13%, less than approximately 12%, less than approximately 11%, less than approximately 10%, less than approximately 9%, less than approximately 8%, less than approximately 7%, less than approximately 6%, less than approximately 5%, less than approximately 4%, less than approximately 3%, less than approximately 2%, or less than approximately 1%) contain modified nucleotides.

[0172] In other embodiments, approximately 1% to approximately 25% of the nucleotides within the double-stranded region of siRNA (e.g., approximately 1% to 25%, 2% to 25%, 3% to 25%, 4% to 25%, 5% to 25%, 6% to 25%, 7% to 25%, 8% to 25%, 9% to 25%, 10% to 25%, 11% to 25%, 12% to 25%, 13% to 25%, 14% to 25%, 15% to 25%, 16% to 25%, 17% to 25%, 18% to 25%, 19% to 25%, 20% to 25%, 21% to 25%, 22% to 25%, 23% to 25%, 24% to 25%, etc.) or approximately 1% to approximately 20% (e.g., approximately 1% to 20%). %, 2%~20%, 3%~20%, 4%~20%, 5%~20%, 6%~20%, 7%~20%, 8%~20%, 9%~20%, 10%~20%, 11%~20%, 12%~20%, 13%~20%, 14%~20%, 15%~20%, 16%~20%, 17%~20%, 18 %~20%, 19%~20%, 1%~19%, 2%~19%, 3%~19%, 4%~19%, 5%~19%, 6%~19%, 7%~19%, 8%~19%, 9%~19%, 10%~19%, 11%~19%, 12%~19%, 13%~19%, 14%~19%, 15%~19%, 16%~19%, 17%~19%, 18%~19%, 1%~18%, 2%~18%, 3%~18%, 4%~18%, 5%~18%, 6%~18%, 7%~18%, 8%~18%, 9%~18%, 10%~18%, 11%~18%, 12%~18%, 13%~18%, 14%~1 8%, 15%~18%, 16%~18%, 17%~18%, 1%~17%, 2%~17%, 3%~17%, 4%~17%, 5%~17%, 6%~17%, 7%~17%, 8%~17%, 9%~17%, 10%~17%, 11%~17%, 12%~17%, 13%~17%, 14% (e.g., ~17%, 15%~17%, 16%~17%, 1%~16%, 2%~16%, 3%~16%, 4%~16%, 5%~16%, 6%~16%, 7%~16%, 8%~16%, 9%~16%, 10%~16%, 11%~16%, 12%~16%, 13%~16%, 14%~16%, 15%~16%, 1%~15%, 2%~15%, 3%~15%, 4%~15%, 5%~15%, 6%~15%, 7%~15%, 8%~15%, 9%~15%, 10%~15%, 11%~15%, 12%~15%, 13%~15%, 14%~15%, etc.) include modified nucleotides.

[0173] In a further embodiment, for example, when one or both strands of the siRNA are selectively modified in uridine and / or guanosine nucleotides, the resulting modified siRNA may have less than about 30% modified nucleotides (e.g., less than about 30%, less than about 29%, less than about 28%, less than about 27%, less than about 26%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% modified nucleotides), or from about 1% to about 30% modified nucleotides (e.g., from about 1% to 30%, 2% to 30%, 3% to 30%, 4% to 30%, 5% to 30%, 6% to 30%, 7% to 30%, 8% to 30%, 9% to 30%, 10% to 30%, 11% to 30%, 12% to 30%, 13% to 30%, 14% to 30%, 15% to 30%, 16% to 30%, 17% to 30%, 18% to 30%, 19% to 30%, 20% to 30%, 21% to 30%, 22% to 30%, 23% to 30%, 24% to 30%, 25% to 30%, 26% to 30%, 27% to 30%, 28% to 30%, or 29% to 30% modified nucleotides).

[0174] Selection of siRNA sequences Suitable siRNA sequences can be identified using any means known in the art. Typically, the methods described in Elbashir et al., Nature, 411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001) are combined with the rational design rules described in Reynolds et al., Nature Biotech., 22(3):326-330 (2004).

[0175] Generally, the nucleotide sequence 3' of the AUG start codon of the transcript from the target gene of interest is scanned for dinucleotide sequences (e.g., AA, NA, CC, GG, or UU (N=C, G, or U)) (see, e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). Nucleotides adjacent to 3' of the dinucleotide sequence are identified as potential siRNA sequences (i.e., target sequences or sense strand sequences). Typically, 19, 21, 23, 25, 27, 29, 31, 33, 35 or more nucleotides adjacent to 3' of the dinucleotide sequence are identified as potential siRNA sequences. In some embodiments, the dinucleotide sequence is an AA or NA sequence, and 19 nucleotides adjacent to 3' of the AA or NA dinucleotide are identified as potential siRNA sequences. siRNA sequences are usually spaced apart at different positions along the length of the target gene. To further enhance the silencing efficiency of siRNA sequences, potential siRNA sequences may be analyzed to identify regions that do not contain homologous areas with other coding sequences, for example, in target cells or organisms. For example, a suitable siRNA sequence of approximately 21 base pairs typically does not have more than 16-17 consecutive base pairs homologous to the coding sequence in the target cell or organism. When expressing siRNA sequences from an RNA Pol III promoter, siRNA sequences lacking more than 4 consecutive A or T bases are selected.

[0176] Once potential siRNA sequences are identified, complementary sequences (i.e., antisense strand sequences) can be designed. Potential siRNA sequences can also be analyzed using various criteria known in the art. For example, to improve their silencing efficiency, siRNA sequences may be analyzed by rational design algorithms to identify sequences having one or more of the following characteristics: (1) G / C content of approximately 25% to 60% G / C, (2) at least three A / U at positions 15-19 of the sense strand, (3) no internal repeats, (4) A at position 19 of the sense strand, (5) A at position 3 of the sense strand, (6) U at position 10 of the sense strand, (7) not G / C at position 19 of the sense strand, and (8) not G at position 13 of the sense strand. siRNA design tools that incorporate algorithms useful for siRNA selection, assigning appropriate values ​​to each of these characteristics, can be found, for example, at http: / / boz094.ust.hk / RNAi / siRNA. Those skilled in the art will understand that sequences possessing one or more of the aforementioned characteristics may be selected as potential siRNA sequences for further analysis and testing.

[0177] Furthermore, potential siRNA sequences possessing one or more of the following criteria can often be excluded as siRNA: (1) sequences containing regions with four or more identical bases in a row; (2) sequences containing G homopolymers (i.e., to reduce the possibility of nonspecific action due to the structural properties of these polymers); (3) sequences containing triple base motifs (e.g., GGG, CCC, AAA, or TTT); (4) sequences containing regions with seven or more G / C units in a row; and (5) sequences containing four or more direct repeats of bases within the candidate that result in an internal foldback structure. However, those skilled in the art will understand that sequences possessing one or more of the aforementioned features can still be selected as potential siRNA sequences for further analysis and testing.

[0178] In some embodiments, potential siRNA sequences can be further analyzed based on siRNA double-strand asymmetry, as described, for example, in Khvorova et al., Cell, 115:209-216 (2003) and Schwarz et al., Cell, 115:199-208 (2003). In other embodiments, potential siRNA sequences can be further analyzed based on secondary structure at the target site, as described, for example, in 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 http: / / www.bioinfo.rpi.edu / applications / mfold / rna / form1.cgi) to select siRNA sequences favorable to accessibility at the target site, with a low presence of secondary structures in the form of base pairings and stem-loops.

[0179] After potential siRNA sequences are identified, they can be analyzed for the presence of any immunostimuli, for example, using in vitro cytokine assays or in vivo animal models. Motifs within the sense and / or antisense strands 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 indicators of whether the sequence may be immunostimulant. If an siRNA molecule is found to be immunostimulant, it can then be modified to reduce its immunostimulant properties, as described herein. As a non-limiting example, to determine whether an siRNA is immunostimulant or non-immunostimulant, the siRNA sequence can be brought into contact with mammalian responder cells under conditions that produce a detectable immune response. Mammalian responder cells may be from naive mammals (i.e., mammals that have not previously come into contact with the gene product of the siRNA sequence). Mammalian responder cells may be, for example, peripheral blood mononuclear cells (PBMCs), macrophages, etc. Detectable immune responses may include the production of cytokines or growth factors, such as TNF-α, IFN-α, IFN-β, IFN-γ, IL-6, IL-12, and combinations thereof. siRNA molecules identified as immunostimulant can then be modified to reduce their immunostimulant properties by substituting at least one nucleotide of the sense strand and / or antisense strand with a modified nucleotide. For example, less than 30% of the nucleotides in the double-stranded region of an siRNA double helix (e.g., less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%) can be replaced with a modified nucleotide such as a 2'OMe nucleotide. The modified siRNA can then be brought into contact with mammalian responder cells as described above to confirm that its immunostimulant properties are reduced or suppressed.

[0180] Suitable in vitro methods for detecting immune responses include the dual monoclonal antibody sandwich immunoassay method by David et al. (U.S. Patent No. 4,376,110), the monoclonal-polyclonal antibody sandwich assay (Wide et al., in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970)), the "Western blot" method by Gordon et al. (U.S. Patent No. 4,452,901), immunoprecipitation of labeled ligands (Brown et al., J. Biol. Chem., 255:4980-4983 (1980)), enzyme-linked immunosorbent assay (ELISA) as described by, for example, Raines et al., J. Biol. Chem., 257:5154-5160 (1982), and immunocytochemistry methods including the use of fluorescent dyes (Brooks et al. This includes, but is not limited to, the immunoassays described above, including those described in U.S. Patent Nos. 3,817,827, 3,850,752, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, and 4,098,876. The disclosures of these references are incorporated herein by reference in their entirety for all purposes.

[0181] Non-limiting examples of in vivo models for detecting immune responses include, for example, the in vivo mouse cytokine induction assay described in Judge et al., Mol. Ther., 13:494-505 (2006). In certain embodiments, the assays that can be performed are as follows: (1) siRNA can be administered into the lateral tail vein by standard intravenous injection; (2) blood can be collected by cardiac puncture approximately 6 hours after administration and processed as plasma for cytokine analysis; and (3) cytokines can be quantified using a sandwich ELISA kit according to the manufacturer's instructions (e.g., mouse and human IFN-α (PBL Biomedical; Piscataway, NJ), human IL-6 and TNF-α (eBioscience; San Diego, Calif.), and mouse IL-6, TNF-α, and IFN-γ (BD Biosciences; San Diego, Calif.)).

[0182] Monoclonal antibodies that specifically bind to cytokines and growth factors are commercially available from multiple sources and can be prepared using methods known in the art (see, for example, Kohler et al., Nature, 256:495-497 (1975) and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publication, New York (1999)). The preparation of monoclonal antibodies has been previously described and can be carried out by any means known in the art (Buhring et al., in Hybridoma, Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, monoclonal antibodies are labeled (e.g., with any composition detectable by spectroscopic, photochemical, biochemical, electrical, optical, or chemical means) to facilitate detection.

[0183] Production of siRNA molecules siRNA can be provided in several forms, including, for example, as one or more isolated small interfering RNA (siRNA) double helixs, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a transcription cassette in a DNA plasmid. siRNA sequences may have overhangs (e.g., 3' or 5' overhangs as described in Elbashir et al., Genes Dev., 15:188 (2001) or Nykanen et al., Cell, 107:309 (2001)) or they may lack overhangs (i.e., have blunt ends).

[0184] Long precursor RNAs can be obtained using RNA populations, or siRNAs can be constructed using long precursor RNAs that have substantial or complete identity with respect to a selected target sequence. RNAs can be isolated, synthesized, and / or cloned from cells or tissues according to methods well known to those skilled in the art. RNAs can be mixed populations (obtained from cells or tissues, transcribed from cDNA, subtracted, selected, etc.) or they can represent a single target sequence. RNAs 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 cloned cDNA), or chemically synthesized.

[0185] To form long dsRNAs, in the case of synthetic RNA, the complement is also transcribed in vitro and hybridized to form the dsRNA. When using naturally occurring RNA populations, the RNA complement is also provided (for example, to form dsRNAs for digestion by E. coli RNAse III or Dicer) by, for example, transcribing the cDNA corresponding to the RNA population or by using RNA polymerase. The precursor RNA is then hybridized to form the double-stranded RNA to be digested. The dsRNA can be administered directly to the target or digested in vitro before administration.

[0186] Methods for RNA isolation, RNA synthesis, nucleic acid hybridization, cDNA library preparation and screening, and PCR are well known in the art (see, for example, Gubler and Hoffman, Gene, 25:263-269 (1983), Sambrook et al. (cited above), and Ausubel et al. (cited above)), and the PCR method is also well known (see, for example, U.S. Patent Nos. 4,683,195 and 4,683,202, and PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Expression libraries are also well known to those skilled in the art. Further basic documents disclosing general uses in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989), Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990), and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994). The disclosures of these references are incorporated herein by reference in their entirety for all purposes.

[0187] Preferably, the siRNA is chemically synthesized. Oligonucleotides containing the siRNA molecule of the present invention can be synthesized using any of the various 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). The synthesis of oligonucleotides uses common nucleic acid protecting groups and coupling groups, such as dimethoxytrityl at the 5' terminus and phosphoramidite at the 3' terminus. As a non-limiting example, small-scale synthesis can be carried out using an Applied Biosystems synthesizer using a 0.2 μmol scale protocol. Alternatively, synthesis on a 0.2 μmol scale can be carried out using a Protogene (Palo Alto, Calif.) 96-well plate synthesizer. However, synthesis on larger or smaller scales is also within the scope of this invention. Suitable reagents for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those skilled in the art.

[0188] siRNA molecules can also be synthesized by tandem synthesis techniques, where both strands are synthesized as fragments or chains of a single continuous oligonucleotide separated by a cleavable linker, which is then cleaved to obtain separate fragments or chains that hybridize to form an siRNA double helix. The linker can be a polynucleotide linker or a non-nucleotide linker. Tandem synthesis of siRNA can be readily adapted to both multi-well / multi-plate synthesis platforms and large-scale synthesis platforms using batch reactors, synthesis columns, etc. Alternatively, an siRNA molecule can be constructed from two separate oligonucleotides, where one oligonucleotide contains the sense strand of the siRNA and the other contains the antisense strand. For example, each strand can be synthesized separately and then joined together by hybridization or ligation after synthesis and / or deprotection. In other specific cases, an siRNA molecule can be synthesized as a single fragment of a continuous oligonucleotide, where the self-complementary sense and antisense regions hybridize to form an siRNA double helix with a hairpin secondary structure.

[0189] Modification of siRNA sequences In certain embodiments, the siRNA molecule comprises a double helix having two strands and at least one modified nucleotide in the double-stranded region, where each strand is approximately 15 to 60 nucleotides long. Advantageously, the modified siRNA is less immunostimulant than the corresponding unmodified siRNA sequence but retains the ability to silence the expression of the target sequence. In preferred embodiments, the degree of chemical modification introduced into the siRNA molecule balances the reduction or suppression of the siRNA's immunostimulant properties with the retention of RNAi activity. As a non-limiting example, an siRNA molecule targeting a target gene may be minimally modified in selective uridine and / or guanosine nucleotides within the siRNA double helix (e.g., less than approximately 30%, less than approximately 25%, less than approximately 20%, less than approximately 15%, less than approximately 10%, or less than approximately 5%) to eliminate the immune response induced by the siRNA while retaining its ability to silence the target gene expression.

[0190] Examples of modified nucleotides suitable for use in the present invention 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 Saenger, Principles of Nucleic Acid Structure, Springer-Verlag Ed. (1984), are also suitable for use in siRNA molecules. Such modified nucleotides include, but are not limited to, locked nucleic acid (LNA) nucleotides (e.g., 2'-O,4'-C-methylene-(D-ribofuranosyl)nucleotide), 2'-O-(2-methoxyethyl)(MOE) nucleotide, 2'-methyl-thio-ethyl nucleotide, 2'-deoxy-2'-fluoro(2'F) nucleotide, 2'-deoxy-2'-chloro(2'Cl) nucleotide, and 2'-azido nucleotide. In certain cases, the siRNA molecules described herein include one or more G-clamp nucleotides. A G-clamp nucleotide refers to a modified cytosine analog in which the modification confers the ability to hydrogen bond to both the Watson-Crick and Hoogsteen faces of a complementary guanine nucleotide in the double helix (see, e.g., Lin et al., J.Am.Chem.Soc., 120:8531-8532 (1998)). Furthermore, nucleotides having nucleotide base analogs, such as C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamide, and nitroazole derivatives, such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, for example, Loakes, Nucl. Acids Res., 29:2437-2447 (2001)), can be incorporated into the siRNA molecule.

[0191] In certain embodiments, an siRNA molecule may further include one or more chemical modifications, such as terminal cap portions, phosphate backbone modifications, etc. Examples of terminal cap portions include inverted deoxydebase residues, glyceryl modifications, 4',5'-methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threopentofuranosyl nucleotides, acyclic 3',4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide portions, 3'-3'-inverted debase portions, 3'-2'-inverted nucleotide portions, 3'-2'-inverted debase portions, 5'-5'-inverted nucleotide portions, 5'-5'-inverted debase portions, This includes, but is not limited to, 3'-5'-reverse deoxydebase moieties, 5'-amino-alkyl phosphates, 1,3-diamino-2-propyl phosphates, 3-aminopropyl phosphates, 6-aminohexyl phosphates, 1,2-aminododecyl phosphates, hydroxypropyl phosphates, 1,4-butanediol phosphates, 3'-phosphoromidates, 5'-phosphoromidates, hexyl phosphates, aminohexyl phosphates, 3'-phosphates, 5'-amino, 3'-phosphorothioate, 5'-phosphorothioate, phosphorodithioate, and crosslinked or uncrosslinked methylphosphonates or 5'-mercapto moieties (see, for example, U.S. Patent No. 5,998,203, Beaucage et al., Tetrahedron 49:1925 (1993)).Non-limiting examples of phosphate backbone modifications (i.e., resulting in modified internucleotide linkages) include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitution (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)). Such chemical modifications can occur at the 5' end and / or 3' end of the sense strand, antisense strand, or both strands of the siRNA. The disclosures of these references are hereby incorporated by reference in their entirety for all purposes.

[0192] In some embodiments, the sense strand and / or antisense strand of the siRNA molecule can further comprise a 3' end overhang having from about 1 to about 4 (e.g., 1, 2, 3, or 4) 2'-deoxyribonucleotides and / or any combination of modified and unmodified nucleotides. Further examples of modified nucleotides and types of chemical modifications that can be introduced into siRNA molecules are described, for example, in UK Patent No. GB2,397,818B and U.S. Patent Publications Nos. 20040192626, 20050282188, and 20070135372, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

[0193] The siRNA molecules described herein may optionally contain one or more non-nucleotides in one or both strands of the siRNA. As used herein, the term “non-nucleotide” refers to any group or compound, including sugar and / or phosphate substitutions, that can be incorporated into a nucleic acid chain in place of one or more nucleotide units, allowing the remaining bases to exhibit their activity. This group or compound does not contain any commonly recognized nucleotide bases such as adenosine, guanine, cytosine, uracil, or thymine, and is therefore debasic in that it lacks a base at the 1' position.

[0194] In other embodiments, the chemical modification of siRNA includes attaching a conjugate to the siRNA molecule. The conjugate may be attached to the 5' and / or 3' ends of the sense and / or antisense strands of the siRNA via covalent bonds, such as a biodegradable linker. The conjugate may also be attached to the siRNA via, for example, a carbamate group or other linking group (see, for example, U.S. Patent Publications 20050074771, 20050043219, and 20050158727). In certain cases, the conjugate is a molecule that facilitates the delivery of siRNA to cells. Examples of conjugate molecules suitable for binding to siRNA include, but are not limited to, 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 their derivatives), sugars (e.g., galactose, galactosamine, N-acetylgalactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands for cell receptors capable of mediating cell uptake, and combinations thereof (see, for example, U.S. Patent Publications 20030130186, 20040110296, and 20040249178, and U.S. Patent No. 6,753,423). Other examples include lipophilic moieties, vitamins, polymers, peptides, proteins, nucleic acids, small molecules, oligosaccharides, carbohydrate clusters, intercalators, sub-groove binders, cleavage agents, and crosslinking agent conjugate molecules, as described in U.S. Patent Publication Nos. 20050119470 and 20050107325. Further examples include 2'-O-alkylamines, 2'-β-alkoxyalkylamines, polyamines, C5-cationically modified pyrimidines, cationic peptides, guanidium groups, amidininium groups, and cationic amino acid conjugate molecules, as described in U.S. Patent Publication Nos. 20050153337.Additional examples include hydrophobic groups, membrane-active compounds, cell-permeable compounds, cell-targeting signals, interaction modifiers, and steric stabilizer conjugate molecules described in U.S. Patent Publication No. 20040167090. Further examples include conjugate molecules described in U.S. Patent Publication No. 20050239739. The type of conjugate used and the degree of conjugation with the siRNA molecule can be evaluated for improved siRNA pharmacokinetic profile, bioavailability, and / or stability while maintaining RNAi activity. Thus, those skilled in the art can screen siRNA molecules conjugated with various conjugates using any of the various well-known in vitro cell cultures or in vivo animal models to identify those with improved properties and full RNAi activity. The disclosures of the above patent documents are incorporated herein by reference in their entirety for all purposes.

[0195] target genes In certain embodiments, the nucleic acid component of the lipid nanoparticles described herein (e.g., siRNA) can be used to downregulate or silencing the translation (i.e., expression) of a target gene. Target genes include, but are not limited to, genes related to viral infection and survival, genes related to metabolic diseases and disorders (e.g., liver diseases and liver damage), genes related to tumorigenesis and cell transformation (e.g., cancer), angiogenic genes, immunomodulatory genes such as those related to inflammatory and autoimmune responses, ligand receptor genes, and genes related to neurodegenerative disorders. In certain embodiments, the target gene is expressed in hepatocytes.

[0196] Genes related to viral infection and survival include those expressed by the virus to bind to, invade, and replicate within cells. Of particular interest are viral sequences associated with chronic viral diseases. In particular, the target viral sequences include filoviruses such as Ebola virus and Marburg virus (see, for example, Geisbert et al., J. Infect. Dis., 193:1650-1657 (2006)), arenaviruses such as Lassa virus, Junin virus, Machupo virus, Guanalitovirus, and Sabia virus (Buchmeier et al., Arenaviridae: the viruses and their replication, In: FIELDS VIROLOGY, Knipe et al. (eds.), 4th ed., Lippincott-Raven, Philadelphia, (2001)), and influenza viruses such as influenza A, B, and C viruses (see, for example, Steinhauer et al., Annu Rev Genet., 36:305-332 (2002) and Neumann et al., J Gen See Virol., 83:2635-2662 (2002), hepatitis viruses (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. Natl. Acad. Sci. USA, 100:2783 (2003), Kapadia et al., Proc. Natl. Acad. Sci. USA, 100:2014 (2003), and FIELDS VIROLOGY, Knipe et al. (eds.), 4th See ed., Lippincott-Raven, Philadelphia (2001), human immunodeficiency virus (HIV) (Banerjea et al., Mol. Ther., 8:62 (2003), Song et al., J. Virol.This includes sequences from 77:7174 (2003), Stephenson, JAMA, 289:1494 (2003), Qin et al., Proc. Natl. Acad. Sci. USA, 100:183 (2003), herpesvirus (Jia et al., J. Virol., 77:3301 (2003)), and human papillomavirus (HPV) (Hall et al., J. Virol., 77:6066 (2003), Jiang et al., Oncogene, 21:6041 (2002)).

[0197] 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-bound proteins (e.g., VP40, glycoprotein (GP), VP24). The complete genome sequence of the Ebola virus is described, for example, in Genbank accessions NC_002549, AY769362, NC_006432, NC_004161, AY729654, AY354458, AY142960, AB050936, AF522874, AF499101, AF272001, and AF086833. The sequence of Ebola virus VP24 is described, for example, in Genbank accessions U77385 and AY058897. The sequence of Ebola virus L-pol is described, for example, in Genbank accession number X67110. The sequence of Ebola virus VP40 is described, for example, in Genbank accession number AY058896. The sequence of Ebola virus NP is described, for example, in Genbank accession number AY058895. The sequence of Ebola virus GP is described, for example, in Genbank accession number AY058898, Sanchez et al., Virus Res., 29:215-240 (1993), Will et al., J. Virol., 67:1203-1210 (1993), Volchkov et al., FEBS Lett., 305:181-184 (1992), and U.S. Patent No. 6,713,069. Further Ebola virus sequences are described, for example, in Genbank accession numbers L11365 and X61274. The complete genome sequence of Marburg virus is described, for example, in Genbank accession numbers NC_001608, AY430365, AY430366, and AY358025. The sequence of Marburg virus GP is described, for example, in Genbank accession numbers AF005734, AF005733, and AF005732. The sequence of Marburg virus VP35 is described, for example, in Genbank accession numbers AF005731 and AF005730.Further Marburg virus sequences are described, for example, in Genbank accession numbers X64406, Z29337, AF005735, and Z12132. Non-limiting examples of siRNA molecules targeting Ebola virus and Marburg virus nucleic acid sequences include those described in U.S. Patent Publication 20070135370, the disclosure of which is incorporated herein in its entirety by reference for any purpose.

[0198] Exemplary influenza virus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences encoding nucleoproteins (NP), matrix proteins (M1 and M2), non-structural proteins (NS1 and NS2), RNA polymerases (PA, PB1, PB2), neuraminidase (NA), and hemagglutinin (HA). Examples of influenza A NP sequences include Genbank accession numbers NC_004522, AY818138, AB166863, AB188817, AB189046, AB189054, AB189062, AY646169, AY646177, AY651486, AY651493, AY651494, AY651495, AY651496, AY This is described in 651497, AY651498, AY651499, AY651500, AY651501, AY651502, AY651503, AY651504, AY651505, AY651506, AY651507, AY651509, AY651528, AY770996, AY790308, AY818138, and AY818140. Examples of PA sequences for influenza A include Genbank accession numbers AY818132, AY790280, AY646171, AY818132, AY818133, AY646179, AY818134, AY551934, AY651613, AY651610, AY651620, AY651617, AY651600, and AY6 These are described in 51611, AY651606, AY651618, AY651608, AY651607, AY651605, AY651609, AY651615, AY651616, AY651640, AY651614, AY651612, AY651621, AY651619, AY770995, and AY724786. Non-limiting examples of siRNA molecules targeting the nucleic acid sequence of the influenza virus include those described in U.S. Patent Publication 20070218122, the disclosure of which is incorporated herein in its entirety by reference for any purpose.

[0199] Exemplary hepatitis virus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences that encode transcription and translation-related nucleic acid sequences (e.g., En1, En2, X, P), as well as structural proteins (e.g., core proteins including C protein and C-related proteins, capsid and envelope proteins including S, M, and / or L proteins, or fragments thereof) (see, for example, FIELDS VIROLOGY (above)). Exemplary hepatitis C virus (HCV) nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences that encode the 5' untranslated region (5'UTR), 3' untranslated region (3'UTR), polyprotein translation start codon region, internal ribosome entry site (IRES) sequence, and / or core proteins, E1 protein, E2 protein, p7 protein, NS2 protein, NS3 protease / helicase, NS4A protein, NS4B protein, NS5A protein, and / or NS5B RNA-dependent RNA polymerase. The HCV genome sequence is described, for example, in Genbank accession numbers NC_004102 (HCV genotype 1a), AJ238799 (HCV genotype 1b), 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). The nucleic acid sequence of the hepatitis A virus is, for example, listed under Genbank accession number NC_001489; the nucleic acid sequence of the hepatitis B virus is, for example, listed under Genbank accession number NC_003977; the nucleic acid sequence of the hepatitis D virus is, for example, listed under Genbank accession number NC_001653; the nucleic acid sequence of the hepatitis E virus is, for example, listed under Genbank accession number NC_001434; and the nucleic acid sequence of the hepatitis G virus is, for example, listed under Genbank accession number NC_001710. Silencing sequences encoding genes related to viral infection and survival can be conveniently used in combination with the administration of conventional drugs used to treat viral conditions.Non-limiting examples of siRNA molecules targeting hepatitis virus nucleic acid sequences include those described in U.S. Patent Publications 20060281175, 20050058982, and 20070149470, U.S. Patent No. 7,348,314, and U.S. Provisional Patent Application No. 61 / 162,127 filed on March 20, 2009, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0200] Genes associated with metabolic diseases and disorders (e.g., liver-targeted disorders, as well as liver diseases and liver damage) include, for example, genes expressed in dyslipidemia (e.g., liver X receptors such as LXRα and LXRβ (Genbank accession number NM_007121), farnesoid X receptor (FXR) (Genbank accession number NM_005123), sterol regulatory element binding protein (SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG coenzyme-A reductase), apolipoprotein B (ApoB) (Genbank accession number NM_000384), apolipoprotein CIII (ApoC3) (Genbank accession numbers NM_000040 and NG_008949) This includes REGION:5001.8164), and apolipoprotein E (ApoE) (Genbank accession numbers NM_000041 and NG_007084 REGION:5001.8612), as well as diabetes (e.g., glucose 6-phosphatase) (e.g., Forman et al., Cell, 81:687 (1995), Seol et al., Mol. Endocrinol., 9:72 (1995), Zavacki et al., Proc. Natl. Acad. Sci. USA, 94:7909 (1997), Sakai et al., Cell, 85:1037-1046 (1996), Duncan et al., J. Biol. Chem., 272:12778-12785 (1997), Willy et al., Genes See Dev., 9:1033-1045 (1995), Lehmann et al., J. Biol. Chem., 272:3137-3140 (1997), Janowski et al., Nature, 383:728-731 (1996), and Peet et al., Cell, 93:693-704 (1998). Those skilled in the art will understand that genes associated with metabolic diseases and disorders (e.g., disorders targeting the liver, as well as liver diseases and liver disorders) include genes expressed in the liver itself as well as genes expressed in other organs and tissues.Silencing sequences encoding genes associated with metabolic diseases and disorders can be conveniently used in combination with the administration of conventional drugs used to treat such diseases or disorders. Non-limiting examples of siRNA molecules targeting the ApoB gene include those described in U.S. Patent Publication 20060134189, which is incorporated herein by reference in its entirety for all purposes. Non-limiting examples of siRNA molecules targeting the ApoC3 gene include those described in U.S. Provisional Patent Application 61 / 147,235, filed on 26 January 2009, which is incorporated herein by reference in its entirety for all purposes.

[0201] Examples of gene sequences associated with tumorigenesis and cell transformation (e.g., cancer or other neoplasm formation) include: mitotic kinesins such as Eg5 (KSP, KIF11; Genbank accession number NM_004523); serine / threonine kinases such as polo-like kinase 1 (PLK-1) (Genbank accession number NM_005030, Barr et al., Nat. Rev. Mol. Cell. Biol., 5:429-440 (2004)); tyrosine kinases such as WEE1 (Genbank accession numbers NM_003390 and NM_001143976); apoptosis inhibitors such as XIAP (Genbank accession number NM_001167); and CSN1, CSN2, CSN3, CSN4, CSN5 (JAB1; Genbank accession number NM_006837) These include COP9 signalosome subunits such as CSN6, CSN7A, CSN7B, and CSN8; ubiquitin ligases such as COP1 (RFWD2; Genbank accession numbers NM_022457 and NM_001001740); and histone deacetylases such as HDAC1, HDAC2 (Genbank accession number NM_001527), HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, and HDAC9. Non-limiting examples of siRNA molecules targeting the Eg5 and XIAP genes are those described in U.S. Patent Application No. 11 / 807,872, filed on May 29, 2007, which is incorporated herein by reference in its entirety for all purposes. Non-limiting examples of siRNA molecules targeting the PLK-1 gene are described in U.S. Patent Publications 20050107316 and 20070265438, and U.S. Patent Application 12 / 343,342, filed December 23, 2008, the disclosures of which are incorporated herein by reference in their entirety for any purpose. Non-limiting examples of siRNA molecules targeting the CSN5 gene include those described in U.S. Provisional Patent Application 61 / 045,251, filed April 15, 2008, the disclosures of which are incorporated herein by reference in their entirety for any purpose.

[0202] Further examples of gene sequences associated with tumorigenesis and cell transformation include translocation sequences, e.g., MLL fusion genes, BCR-ABL (Wilda et al., Oncogene, 21:5716 (2002), Scherr et al., Blood, 101:1566 (2003)), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO, and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003)); overexpression sequences, e.g., multidrug resistance genes (Nieth et al., FEBS Lett., 545:144 (2003), Wu et al., Cancer Res. 63:1515 (2003)), cyclins (Li et al., Cancer Res. 63:3593 (2003), Zou et al.) al., Genes Dev., 16:2923 (2002)), beta-catenin (Verma et al., Clin Cancer Res., 9:1291 (2003)), telomerase gene (Kosciolek et al., Mol Cancer Ther., 2:209 (2003)), c-MYC, N-MYC, BCL-2, growth factor receptor (e.g., EGFR / ErbB1 (Genbank accession numbers NM_005228, NM_201282, NM_201283, and NM_201284, also Nagy et al. Exp. Cell) See Res., 285:39-49 (2003), examples include ErbB2 / HER-2 (Genbank accession numbers NM_004448 and NM_001005862), ErbB3 (Genbank accession numbers NM_001982 and NM_001005915), and ErbB4 (Genbank accession numbers NM_005235 and NM_001042599); and variant sequences such as RAS (outlined in Tuschl and Borkhardt, Mol. Interventions, 2:158 (2002)). Non-limiting examples of siRNA molecules targeting the EGFR gene include those described in U.S. Patent Application No. 11 / 807,872, filed on 29 May 2007, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0203] Silencing sequences encoding DNA repair enzymes is used 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, such as integrins, selectins, and metalloproteinases. The examples given above are not exclusive. Those skilled in the art will understand that any whole or partial gene sequence that promotes or accelerates tumorigenesis or cell transformation, tumor growth, or tumor migration may be included as a template sequence.

[0204] Angiogenesis genes can promote the formation of new blood vessels. Of particular interest are vascular endothelial growth factor (VEGF) (Reich et al., Mol.Vis., 9:210 (2003)) or VEGFR. siRNA sequences targeting VEGFR are described, for example, in GB2396864, U.S. Patent Publication No. 20040142895, and CA2456444, the disclosures thereof being incorporated herein by reference in their entirety for all purposes.

[0205] Anti-angiogenic genes can inhibit neoangiogenesis. These genes are particularly useful in the treatment of cancers in which angiogenesis plays a role in the pathological development of the disease. Examples of anti-angiogenic genes include, but are not limited to, endostatins (see, e.g., U.S. Patent No. 6,174,861), angiostatins (see, e.g., U.S. Patent No. 5,639,725), and VEGFR2 (see, e.g., Decaussin et al., J. Pathol., 188:369-377 (1999)), the disclosures thereof, are incorporated herein by reference in their entirety for all purposes.

[0206] Immunomodulatory genes are genes that regulate one or more immune responses. Examples of immunomodulatory genes include, but are not limited to, growth factors (e.g., TGF-α, TGF-β, 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-α, IFN-β, IFN-γ, etc.), and cytokines such as TNF. Fas and Fas ligand genes are also immunomodulatory target sequences of interest (Song et al., Nat.Med., 9:347 (2003)). Genes encoding secondary signaling molecules in hematopoietic cells and lymphoid cells, such as Tec family kinases including Bruton's tyrosine kinase (Btk) (Heinonen et al., FEBS Lett., 527:274 (2002)), are also included in the present invention.

[0207] Cell receptor ligands include ligands that bind to cell surface receptors (e.g., insulin receptor, EPO receptor, G protein-coupled receptor, tyrosine kinase receptor, cytokine receptor, growth factor receptor, etc.) and can regulate (e.g., inhibit, activate, etc.) physiological pathways (e.g., glucose level regulation, hematocytosis, mitosis, etc.) in which those receptors are involved. Examples of cell receptor ligands include, but are not limited to, cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, and G protein-coupled receptor ligands. Templates encoding trinucleotide repeat elongation (e.g., CAG repeat) are used to silence pathogenic sequences in neurodegenerative disorders caused by trinucleotide repeat elongation, such as spinal and bulbar muscular atrophy and Huntington's disease (Caplen et al., Hum. Mol. Genet., 11:175 (2002)).

[0208] Other specific target genes that can be targeted by nucleic acids (e.g., by siRNA) to downregulate or silence gene expression include aortic smooth muscle alpha-2 actin (ACTA2), alcohol dehydrogenase 1A (ADH1A), alcohol dehydrogenase 4 (ADH4), alcohol dehydrogenase 6 (ADH6), afamin (AFM), angiotensinogen (AGT), serine pyruvate aminotransferase (AGXT), and alpha-2-H S-glycoprotein (AHSG), aldo-ketereductase family 1 member C4 (AKR1C4), serum albumin (ALB), alpha-1-microglobulin / bicin precursor (AMBP), angiopoietin-related protein 3 (ANGPTL3), serum amyloid-P component (APCS), apolipoprotein A-II (APOA2), apolipoprotein B-100 (APOB), apolipoprotein C3 (APOC3), apolipoprotein C-IV (APOC4), apolipo Protein F (APOF), beta-2-glycoprotein 1 (APOH), aquaporin-9 (AQP9), bile acid-CoA:amino acid N-acyltransferase (BAAT), C4b-binding protein beta chain (C4BPB), LINC01554 (C5orf27) encoded by an unspecified protein, complement factor 3 (C3), complement factor 5 (C5), complement component C6 (C6), complement component C8 alpha chain (C8A), complement component C8 beta chain (C8B), complement component C8 gamma chain (C8 G) Complement component C9 (C9), calmodulin-binding transcription activator 1 (CAMTA1), CD38 (CD38), complement factor B (CFB), complement factor H-related protein 1 (CFHR1), complement factor H-related protein 2 (CFHR2), complement factor H-related protein 3 (CFHR3), cannabinoid receptor 1 (CNR1), ceruloplasmin (CP), carboxypeptidase B2 (CPB2), connective tissue growth factor (CTGF), CXC motif chemokine 2 (CXCL2), cytochrome P450 1A2 (CYP1A2), cytochrome P450 2A6 (CYP2A6), cytochrome P450 2C8 (CYP2C8), cytochrome P450 2C9 (CYP2C9), cytochrome P450 family 2 subfamily D member 6 (CYP2D6),Cytochrome P450 2E1 (CYP2E1), phylloquinone omega-hydroxylase CYP4F2 (CYP4F2), 7-alpha-hydroxycholest-4-en-3-one 12-alpha-hydroxylase (CYP8B1), dipeptidyl peptidase 4 (DPP4), coagulation factor XII (F12), coagulation factor II (thrombin) (F2), coagulation factor IX (F9), fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), fibrinogen gamma chain (FGG), fibrinogen-like substance 1 (FGL1), flavin-containing monooxygenate FMO3 (Flavin-containing monooxygenase 5), FMO5 (Flavin-containing monooxygenase 5), Group-specific component (vitamin D-binding protein) (GC), Growth hormone receptor (GHR), Glycine N-methyltransferase (GNMT), Hyaluronan-binding protein 2 (HABP2), Hepcidin antimicrobial peptide (HAMP), Hydroxy acid oxidase (glycolate oxidase) 1 (HAO1), HGF activator (HGFAC), Haptoglobin-related proteins; Haptoglobin (HPR), Hemopexin (HPX), Histidine Interalpha-trypsin inhibitor heavy chain H1 (ITIH1), interalpha-trypsin inhibitor heavy chain H2 (ITIH2), interalpha-trypsin inhibitor heavy chain H3 (ITIH3), interalpha-trypsin inhibitor heavy chain H4 (ITIH4), prekallikrein (KLKB1), lactate dehydrogenase A ( LDHA), liver-expressed antimicrobial peptide 2 (LEAP2), leukocyte-derived chemotaxin 2 (LECT2), lipoprotein (a) (LPA), mannan-binding lectin serine peptidase 2 (MASP2), S-adenosylmethionine synthase isoform 1 (MAT1A), NADPH oxidase 4 (NOX4), poly[ADP-ribose] polymerase 1 (PARP1), paraoxonase 1 (PON1), paraoxonase 3 (PON3), vitamin K-dependent protein C (PROC), retinol dehydrogenase 16 (RDH16),Constitutive serum amyloid A4 (SAA4), serine dehydratase (SDS), serpine family A member 1 (SERPINA1), serpine A11 (SERPINA11), calistatin (SERPINA4), corticosteroid-binding globulin (SERPINA6), antithrombin III (SERPINC1), heparin cofactor 2 (SERPIND1), serpine family H member 1 (SERPINH1), solute carrier family 5 member 2 (SLC5A2), sodium / bile acid cotransporter (SLC10A1), solute carrier family 13 member 5 (SLC13A5), solute carrier family 22 member 1 (SLC22A1), solute carrier family 25 member 47 (SLC25A47), solute carrier family 2 facilitating glucose This includes, but is not limited to, transporter member 2 (SLC2A2), sodium-conjugated neutral amino acid transporter 4 (SLC38A4), solute carrier organic anion transporter family member 1B1 (SLCO1B1), sphingomyelin phosphodiesterase 1 (SMPD1), bile salt sulfotransferase (SULT2A1), tyrosine aminotransferase (TAT), tryptophan 2,3-dioxygenase (TDO2), UDP-glucuronosyltransferase 2 family polypeptide B10 (UGT2B10), UDP-glucuronosyltransferase 2 family polypeptide B15 (UGT2B15), UDP-glucuronosyltransferase 2 family polypeptide B4 (UGT2B4), and vitronectin (VTN).

[0209] In addition to their usefulness in silencing the expression of any of the aforementioned genes for therapeutic purposes, the specific nucleic acids (e.g., siRNA) described herein are also useful for research and development applications, as well as for diagnostic, prophylactic, prognostic, clinical, and other healthcare applications. As a non-limiting example, specific nucleic acids (e.g., siRNA) can be used in target validation studies aimed at testing whether a target gene may be a therapeutic target. Specific nucleic acids (e.g., siRNA) can also be used in target identification studies aimed at discovering genes as potential therapeutic targets.

[0210] CRISPR Targeted genome editing has evolved from a niche technology to a method used by many biologists. This evolution has been greatly facilitated by the emergence of CRISPR technology, which involves the repetition of clustered, regularly spaced short palindromic structures (see, for example, Sander et al., Nature Biotechnology, 32(4), 347-355 (2014), International Publications WO2016 / 197132 and WO2016 / 197133, including Supplementary Information). Accordingly, this specification provides improved products (e.g., lipid nanoparticles and their formulations) that can be used in combination with CRISPR technology to treat diseases such as HBV. Regarding targets for CRISPR, guide RNAs (gRNAs) used in CRISPR technology can be designed to target specifically identified sequences, such as target genes (e.g., target genes in the HBV genome). Examples of such target sequences are shown in International Publication WO2016 / 197132. Furthermore, International Publication No. WO2013 / 151665 (see, for example, Table 6; this document is specifically incorporated by reference, including Table 6 and the accompanying sequence listings) lists approximately 35,000 mRNA sequences claimed in relation to mRNA expression constructs. Certain embodiments of the present invention utilize CRISPR technology to target the expression of any of these sequences. Certain embodiments of the present invention may also utilize CRISPR technology to target the expression of target genes discussed herein.

[0211] aiRNA Similar to siRNA, asymmetric interfering RNA (aiRNA) can mobilize the RNA-induced silencing complex (RISC) to mediate sequence-specific cleavage of a target sequence between nucleotides 10 and 11 relative to the 5' end of the antisense strand, thereby resulting in effective silencing of various genes in mammalian cells (Sun et al., Nat. Biotech., 26:1379-1382 (2008)). Typically, an aiRNA molecule contains a short RNA double helix with a sense strand and an antisense strand, the double helix containing overhangs at the 3' and 5' ends of the antisense strand. aiRNAs are generally asymmetric because the sense strand is shorter at both ends compared to the complementary antisense strand. In some embodiments, aiRNA molecules can be designed, synthesized, and annealed under conditions similar to those used for siRNA molecules. As a non-limiting example, aiRNA sequences can be selected and constructed using the methods described above for selecting siRNA sequences.

[0212] In another embodiment, aiRNA double helices of varying lengths (e.g., about 10–25, 12–20, 12–19, 12–18, 13–17, or 14–17 base pairs, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 14–17 base pairs) can be designed to target a target mRNA of interest, including overhangs at the 3' and 5' ends of the antisense strand. In a particular case, the sense strand of the aiRNA molecule is about 10–25, 12–20, 12–19, 12–18, 13–17, or 14–17 nucleotides long, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides long. In other specific cases, the antisense strand of the aiRNA molecule is about 15–60, 15–50, or 15–40 nucleotides long, more typically about 15–30, 15–25, or 19–25 nucleotides long, preferably about 20–24, 21–22, or 21–23 nucleotides long.

[0213] In some embodiments, the 5' antisense overhang contains one, two, three, four, or more non-target nucleotides (e.g., "AA", "UU", "dTdT", etc.). In other embodiments, the 3' antisense overhang contains one, two, three, four, or more non-target nucleotides (e.g., "AA", "UU", "dTdT", etc.). In certain embodiments, the aiRNA molecule described herein may contain, for example, one or more modified nucleotides in the double-stranded (double-stranded) region and / or in the antisense overhang. As a non-limiting example, the aiRNA sequence may contain one or more of the modified nucleotides described above for the siRNA sequence. In preferred embodiments, the aiRNA molecule contains, for example, 2'OMe nucleotides such as 2'OMe-guanosine nucleotide, 2'OMe-uridine nucleotide, or a mixture thereof.

[0214] In certain embodiments, the aiRNA molecule may include an antisense strand corresponding to the antisense strand of an siRNA molecule, for example, one of the siRNA molecules described herein. In other embodiments, the aiRNA molecule may be used to silence the expression of any of the target genes described above, such as genes related to viral infection and survival, genes related to metabolic diseases and disorders, genes related to tumorigenesis and cell transformation, angiogenic genes, immunomodulatory genes such as those related to inflammatory and autoimmune responses, ligand receptor genes, and genes related to neurodegenerative diseases.

[0215] miRNA Generally, microRNAs (miRNAs) are single-stranded RNA molecules, approximately 21-23 nucleotides long, that regulate gene expression. While miRNAs are encoded by genes in their source DNA, they are not translated into proteins (non-coding RNAs). Instead, each primary transcript (pri-miRNA) is processed into a short stem-loop structure called pre-miRNA, which is then processed into a functional mature miRNA. Mature miRNA molecules are either partially or fully complementary to one or more messenger RNA (mRNA) molecules, and their primary function is to downregulate gene expression. Identification of miRNA molecules is described, for example, in Lagos-Quintana et al., Science, 294:853-858, Lau et al., Science, 294:858-862, and Lee et al., Science, 294:862-864.

[0216] The genes encoding miRNAs are much longer than the processed mature miRNA molecules. miRNAs are first transcribed as a primary transcript or pri-miRNA with a cap and poly(A) tail, and in the cell nucleus, they are processed into a short stem-loop structure of about 70 nucleotides known as pre-miRNA. This processing is carried out 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)). These pre-miRNAs are then processed into mature miRNAs in the cytoplasm through interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC) (Bernstein et al., Nature, 409:363-366 (2001)). Either the sense or antisense strand of DNA can serve as a template for generating miRNAs.

[0217] When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one is incorporated into the RISC complex. This strand, known as the guide strand, is selected by the Argonaut protein, a catalytically active RNase in the RISC complex, based on the stability of its 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)). After being incorporated into the active RISC complex, the miRNA base pairs with its complementary mRNA molecule, inducing the degradation and / or translational silencing of the target mRNA.

[0218] Mammalian miRNA molecules are typically complementary to a site within the 3' UTR of a target mRNA sequence. In certain cases, miRNA annealing to a target mRNA inhibits protein translation by blocking the protein translation mechanism. In other specific cases, miRNA annealing to a target mRNA promotes cleavage and degradation of the target mRNA through a process similar to RNA interference (RNAi). miRNAs can also target methylation of genomic sites corresponding to the targeted mRNA. Generally, miRNAs function in conjunction with protein complement, collectively known as miRNPs.

[0219] In certain embodiments, 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 long, more typically about 15–30, 15–25, or 19–25 nucleotides long, preferably about 20–24, 21–22, or 21–23 nucleotides long. In certain other embodiments, the miRNA molecule may contain one or more modified nucleotides. As a non-limiting example, a miRNA sequence may contain one or more of the modified nucleotides described above for an siRNA sequence. In preferred embodiments, the miRNA molecule contains, for example, 2'OMe nucleotides such as 2'OMe-guanosine nucleotide, 2'OMe-uridine nucleotide, or a mixture thereof.

[0220] In some embodiments, miRNA molecules may be used to silence the expression of any of the aforementioned target genes, such as genes related to viral infection and survival, genes related to metabolic diseases and disorders, genes related to tumorigenesis and cell transformation, angiogenic genes, immunomodulatory genes such as those related to inflammatory and autoimmune responses, ligand receptor genes, and genes related to neurodegenerative diseases.

[0221] In other embodiments, one or more agents that block the activity of miRNAs targeting the mRNA of interest are administered using lipid particles (e.g., lipid nanoparticles) of the present invention. Examples of blockers include, but are not limited to, sterically blocked oligonucleotides, locked nucleic acid oligonucleotides, and morpholino oligonucleotides. Such blockers may bind directly to the miRNA or the miRNA-binding site on the target mRNA.

[0222] Antisense oligonucleotides In one embodiment, the nucleic acid is an antisense oligonucleotide targeting a target gene or sequence of interest. The terms “antisense oligonucleotide” or “antisense” include oligonucleotides complementary to the target polynucleotide sequence. An antisense oligonucleotide is a single strand of DNA or RNA that is complementary to the selected sequence. An antisense RNA oligonucleotide inhibits the translation of the complementary RNA strand by binding to that RNA. An antisense DNA oligonucleotide can be used to target a specific complementary (coding or non-coding) RNA. Once binding occurs, this DNA / RNA hybrid can be degraded by the enzyme RNase H. In certain embodiments, the antisense oligonucleotide contains about 10 to about 60 nucleotides, more preferably about 15 to about 30 nucleotides. The term also includes antisense oligonucleotides that may not be strictly complementary to the desired target gene. Therefore, the present invention is available when target nonspecific activity is observed in the antisense, or when an antisense sequence containing one or more mismatches with the target sequence is most preferred for a particular use.

[0223] Antisense oligonucleotides have been shown to be effective and targeted inhibitors of protein synthesis and can therefore be used to specifically inhibit protein synthesis by targeted genes. The effectiveness of antisense oligonucleotides for inhibiting protein synthesis is well established. For example, the synthesis of polygalactauronase and muscarinic acetylcholine receptor type 2 is inhibited by antisense oligonucleotides targeting their respective mRNA sequences (see U.S. Patents 5,739,119 and 5,759,829). Furthermore, examples of antisense inhibition have been demonstrated for nucleoprotein cyclins, multidrug resistance genes (MDR1), ICAM-1, E-selectin, STK-1, striatal GABAA receptors, and human EGF (see Jaskulski et al., Science, 240:1544-6 (1988), Vasanthakumar et al., Cancer Commun., 1:225-32 (1989), Penis et al., Brain Res Mol Brain Res., 15;57:310-20 (1998), and U.S. Patents No. 5,801,154, No. 5,789,573, No. 5,718,709, and No. 5,610,288). Furthermore, antisense constructs that can be used to inhibit and treat various abnormal cell proliferations, such as cancer, are also described (see U.S. Patents No. 5,747,470, 5,591,317, and 5,783,683). The disclosures of these references are incorporated herein by reference in their entirety for all purposes.

[0224] Methods for generating antisense oligonucleotides are known in the art and can be readily adapted to generate antisense oligonucleotides targeting any polynucleotide sequence. The selection of an antisense oligonucleotide sequence specific to a given target sequence involves analysis of the selected target sequence, as well as its secondary structure, T mThis is based on the determination of binding energy and relative stability. Antisense oligonucleotides may be selected based on their relative inability to form dimers, hairpins, or other secondary structures that reduce or block specific binding to target mRNA within the host cell. Highly preferred target regions of mRNA include the region of the AUG translation start codon or its vicinity, and sequences substantially complementary to the 5' region of mRNA. These secondary structure analyses and considerations for target site selection can be performed, for example, using OLIGO primer analysis software v.4 (Molecular Biology Insights) and / or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).

[0225] Ribozyme According to another embodiment of the present invention, lipid nanoparticles associate with ribozymes. Ribozymes are RNA-protein complexes having a specific catalytic domain with endonuclease activity (see Kim et al., Proc. Natl. Acad. Sci. USA., 84:8788-92 (1987), and Forster et al., Cell, 49:211-20 (1987)). For example, many ribozymes promote phosphate transfer reactions with high specificity, often cleaving only one of several phosphate esters in an oligonucleotide substrate (see Cech et al., Cell, 27:487-96 (1981), Michel et al., J. Mol. Biol., 216:585-610 (1990), and Reinhold-Hurek et al., Nature, 357:173-6 (1992)). This specificity stems from the requirement that the substrate binds to the internal guide sequence ("IGS") of the ribozyme via a specific base pairing interaction before the chemical reaction.

[0226] Currently, at least six basic types of naturally occurring enzymatic RNA molecules are known. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans under physiological conditions (and thus can cleave other RNA molecules). Generally, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs via a target-binding portion of the enzymatic nucleic acid, which is held in close proximity to the enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes the target RNA, then binds to it via complementary base pairing, and once bound to the correct site, acts enzymatically to cleave the target RNA. Such strategic cleavage of the target RNA causes it to lose its ability to direct the synthesis of the encoded protein. After the enzymatic nucleic acid has bound to its RNA target and cleaved it, it can be released from that RNA to seek another target, and can repeatedly bind to and cleave new targets.

[0227] Enzymatic nucleic acid molecules can be formed, for example, from hammerhead, hairpin, hepatitis delta virus, group I intron, or RNaseP RNA (associated with an RNA guide sequence) or Neurospora VS RNA motifs. Specific examples of hammerhead motifs are described, for example, in Rossi et al., Nucleic Acids Res., 20:4559-65 (1992). Examples of hairpin motifs are described, for example, in EP0360257, Hampel et al., Biochemistry, 28:4929-33 (1989), Hampel et al., Nucleic Acids Res., 18:299-304 (1990), and U.S. Patent No. 5,631,359. Examples of hepatitis delta virus motifs are described, for example, in Perrotta et al., Biochemistry, 31:11843-52 (1992). Examples of RNaseP motifs are described, for example, in Guerrier-Takada et al., Cell, 35:849-57 (1983). Examples of Neurospora VS RNA ribozyme motifs are described, for example, in Saville et al., Cell, 61:685-96 (1990), Saville et al., Proc. Natl. Acad. Sci. USA, 88:8826-30 (1991), and Collins et al., Biochemistry, 32:2795-9 (1993). Examples of group I introns are described, for example, in U.S. Patent No. 4,987,071. An important feature of the enzymatic nucleic acid molecules used in accordance with the present invention is that they have a specific substrate binding site complementary to one or more DNA or RNA regions of a target gene, and that they have a nucleotide sequence within or around their substrate binding site that confers RNA cleavage activity to the molecule. Therefore, ribozyme constructs are not necessarily limited to the specific motifs mentioned herein. The disclosures of these references are incorporated herein by reference in their entirety for all purposes.

[0228] Methods for generating ribozymes targeting arbitrary polynucleotide sequences are known in the art. Ribozymes may be designed, for example, as described in PCT Publications WO93 / 23569 and WO94 / 02595, and synthesized for in vitro and / or in vivo testing as described therein. These PCT Publications are incorporated herein by reference in their entirety for all purposes.

[0229] Ribozyme activity can be optimized by altering the length of the ribozyme-binding arms, or by chemically synthesizing ribozymes having modifications that inhibit their degradation by serum ribonucleases (see, for example, PCT Publications WO92 / 07065, WO93 / 15187, WO91 / 03162, and WO94 / 13688, EP92110298.4, and U.S. Patent No. 5,334,711 (these describe various chemical modifications that can be made to the sugar portion of the enzyme RNA molecule, and these disclosures are each incorporated herein by reference in their entirety for any purpose)), modifications that enhance their efficacy in cells, and removal of stem II bases for shortening RNA synthesis time and reducing chemical requirements).

[0230] Immunostimulatory oligonucleotides The nucleic acids associated with the lipid particles of the present invention may be immunostimulant, including immunostimulant oligonucleotides (ISSs; single-stranded or double-stranded) that can induce an immune response when administered to a target that may be a mammal such as a human. ISSs include, for example, certain palindromic structures resulting in hairpin secondary structures (see Yamamoto et al., J.Immunol., 148:4072-6 (1992)), or CpG motifs, and other known ISS features (e.g., multiple G domains; see PCT Publication WO96 / 11266, the disclosure of which is incorporated herein in its entirety by reference for any purpose).

[0231] Immunostimulatory nucleic acids are considered sequence-nonspecific if they do not need to specifically bind to a target sequence and reduce its expression in order to induce an immune response. Therefore, certain immunostimulatory nucleic acids may contain sequences corresponding to naturally occurring gene or mRNA regions, but they can still be considered sequence-nonspecific immunostimulatory nucleic acids.

[0232] In one embodiment, the immunostimulatory nucleic acid or oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be methylated or unmethylated. In another embodiment, the immunostimulatory nucleic acid comprises at least one CpG dinucleotide having methylated cytosine. In one embodiment, the nucleic acid comprises a single CpG dinucleotide, and the cytosine in this CpG dinucleotide is methylated. In an alternative embodiment, the nucleic acid comprises at least two CpG dinucleotides, and at least one of these CpG dinucleotides is methylated. In a further embodiment, each cytosine in the CpG dinucleotide present in the sequence is methylated. In another embodiment, the nucleic acid comprises multiple CpG dinucleotides, and at least one of these CpG dinucleotides contains methylated cytosine. Examples of immunostimulatory oligonucleotides suitable for use in the compositions and methods of the present invention are described in PCT application PCT / US08 / 88676, PCT publications WO02 / 069369 and WO01 / 15726, U.S. Patent No. 6,406,705, filed December 31, 2008, and Raney et al., J. Pharm. Exper. Ther., 298:1185-92 (2001), the disclosures thereof, respectively, are incorporated herein by reference in their entirety for any purpose. In certain embodiments, the oligonucleotides used in the compositions and methods of the present invention have a phosphodiester ("PO") or phosphorothioate ("PS") skeleton and / or at least one methylated cytosine residue in a CpG motif.

[0233] mRNA Certain embodiments of the present invention provide compositions and methods that can be used to express one or more mRNA molecules in living cells (e.g., cells in the human body). These mRNA molecules encode one or more polypeptides to be expressed in living cells. In some embodiments, these polypeptides are expressed in a diseased organism (e.g., mammals such as humans), and the expression of these polypeptides improves one or more symptoms of a disease. The compositions and methods of the present invention are particularly useful for treating human diseases caused by a deficiency or reduction in the levels of functional polypeptides in the human body. Accordingly, in certain embodiments, the LNP may comprise one or more nucleic acid molecules, such as one or more mRNA molecules (e.g., a cocktail of mRNA molecules).

[0234] In some embodiments, mRNA(s) are completely encapsulated in nucleic acid-lipid particles (e.g., LNPs). With respect to formulations containing mRNA cocktails, different types of mRNA species present in the cocktail (e.g., mRNAs with different sequences) may be co-encapsulated in the same particle, or each type of mRNA species present in the cocktail may be encapsulated in separate particles. mRNA cocktails can be formulated in the particles described herein using a mixture of two or more individual mRNAs (each having a unique sequence) at the same, similar, or different concentrations or molar ratios. In one embodiment, an mRNA cocktail (corresponding to multiple mRNAs with different sequences) is formulated using the same, similar, or different concentrations or molar ratios for each mRNA species, and different types of mRNA are co-encapsulated in the same particle. In another embodiment, each type of mRNA species present in the cocktail is encapsulated in different particles at the same, similar, or different mRNA concentrations or molar ratios, and these particles (each containing a different mRNA payload) are administered separately (e.g., at different time points according to a therapeutic regimen) or combined (e.g., with a pharmaceutically acceptable carrier) and administered together. The particles described herein are serum-stable, resistant to nuclease degradation, and substantially non-toxic to mammals such as humans.

[0235] Modification of mRNA The mRNA used in the embodiment of the present invention may contain one, two, or more than two nucleoside modifications. In some embodiments, the modified mRNA exhibits reduced degradation in the cells into which it is introduced compared to the corresponding unmodified mRNA.

[0236] In some embodiments, the modified nucleosides include pyridine-4-onribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudridine, 5-propynyluridine, 1-propynyl-pseudridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudridine, 5-taurinomethyl-2-thiouridine, and 1-taurinomethyl This includes -4-thio-uridine, 5-methyl-uridine, 1-methyl-1-pseuduridine, 4-thio-1-methyl-1-pseuduridine, 2-thio-1-methyl-1-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine, dihydropseuduridine, 2-thio-dihydrouridine, 2-thio-dihydropseuduridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseuduridine, and 4-methoxy-2-thio-pseuduridine.

[0237] In some embodiments, the modified nucleosides include 5-azacytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpseudoisocytidine, pyrrolocytidine, pyrrolopseudoisocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thiopseudoisocytidine, and 4-thio-1-methyl -Includes pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0238] In other embodiments, the modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N This includes 6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0239] In certain embodiments, the modified nucleoside is 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudruridine. The α-thio-substituted phosphate moiety is provided to confer stability to the RNA polymer via non-natural phosphorothioate backbone binding. Phosphorothioate RNA exhibits increased nuclease resistance and subsequently a longer half-life in the cellular environment. Phosphorothioate-bound nucleic acids are also expected to reduce the innate immune response through relatively weak binding / activation of innate immune molecules in cells.

[0240] In certain embodiments, for example, when precise timing of protein production is desired, it is desirable to degrade the modified nucleic acid introduced into the cell within the cell. Therefore, the present invention provides a modified nucleic acid containing a degradation domain that can act in a directed manner within the cell.

[0241] In other embodiments, the modified nucleosides include inosine, 1-methylinosine, waiosine, waibutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-8-aza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0242] Selective components of modified nucleic acids In further embodiments, the modified nucleic acid may include other optional components that may be beneficial in some embodiments. These optional components include, but are not limited to, untranslated regions, Kosack sequences, intronic nucleotide sequences, internal ribosome entry sites (IRESs), caps, and poly(A) tails. For example, a 5' untranslated region (UTR) and / or a 3' UTR may be provided, either or both of which may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the translatable region. Nucleic acids containing Kosack sequences are also provided.

[0243] Furthermore, nucleic acids containing one or more intronic nucleotide sequences that can be cleaved from the nucleic acid are provided.

[0244] Untranslated area (UTR) The uncoding region (UTR) of a gene is transcribed but not translated. The 5' UTR begins at the transcription start site and extends to the start codon, but does not include the start codon. The 3' UTR, on the other hand, begins immediately after the stop codon and extends to the transcription termination signal. There is growing evidence regarding the regulatory role that UTRs play in the stability and translation of nucleic acid molecules. To increase molecular stability, the regulatory function of UTRs can be incorporated into the mRNA used in this invention. By incorporating specific functions, it is also possible to ensure the downregulation of transcripts in the event of misdirection to undesirable organ sites.

[0245] 5' capping The 5' cap structure of mRNA is involved in nuclear export, increases mRNA stability, and binds to mRNA cap-binding proteins (CBPs). These CBPs, along with poly(A)-binding proteins, associate to form mature circular mRNA species, thereby contributing to the stability and translational capacity of mRNA within cells. The cap also assists in the removal of the 5' proximal intron during mRNA splicing.

[0246] Endogenous mRNA molecules can be capped at their 5' end, creating a 5'-ppp-5'-triphosphate bond between the terminal guanosine cap residue of the mRNA molecule and the transcribed sense nucleotide at the 5' end. This 5'-guanylate cap can then be methylated to produce an N7-methyl-guanylate residue. The ribose sugars of the 5' end and / or pre-terminal transcribed nucleotides of the mRNA can also be optionally 2'-O-methylated. Removal of the 5'-cap by hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules such as mRNA molecules for degradation.

[0247] IRES array mRNA containing an internal ribosome entry site (IRES) is also useful in carrying out the present invention. The IRES may function as a single ribosome binding site or as one of several ribosome binding sites in the mRNA. mRNA containing two or more functional ribosome binding sites may encode several peptides or polypeptides that are independently translated by ribosomes ("multicistronic mRNA"). If mRNA is provided with an IRES, a second translateable region is optionally provided. Examples of IRES sequences that can be used according to the present invention include, but are not limited to, those derived from picornaviruses (e.g., FMDV), plague virus (CFFV), poliovirus (PV), encephalomyocarditis virus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), swine cholera virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV).

[0248] Poly A Tail During RNA processing, a long chain of adenine nucleotides (poly-A tail) can be added to polynucleotides such as mRNA molecules to increase stability. Immediately after transcription, the 3' end of the transcript may be cleaved, releasing a 3' hydroxyl group. Next, poly-A polymerase adds a chain of adenine nucleotides to the RNA. This process, called polyadenylation, adds a poly-A tail that can be 100-250 residues long.

[0249] Generally, the length of a poly-A tail exceeds 30 nucleotides. In another embodiment, the poly-A tail exceeds 35 nucleotides (for example, at least about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2,000, 2,500, and 3,000 nucleotides).

[0250] In this scenario, the polyA tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the modified mRNA. The polyA tail can also be designed as part of the modified nucleic acid to which it belongs. In this scenario, the polyA tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90%, or more of the total length of the modified mRNA or the total length of the modified mRNA minus the polyA tail.

[0251] Synthesis of mRNA molecules Methods for RNA isolation, RNA synthesis, nucleic acid hybridization, cDNA library preparation and screening, and PCR are well known in the art, as are PCR methods (see U.S. Patents No. 4,683,195 and 4,683,202, and PCR Protocols: A Guide to Methods and Applications (Innis et al., eds., 1990)). (See, for example, Gubler and Hoffman, Gene, 25:263-269 (1983), and Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed., 1989)). Expression libraries are also well known to those skilled in the art. Further basic documents disclosing the general uses of the present invention include Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990), and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994). The disclosures of these references are incorporated herein by reference in their entirety for all purposes.

[0252] Coded polypeptide The mRNA components of the lipid nanoparticles described herein can be used to express target polypeptides. Certain diseases in humans are caused by the absence or impairment of functional proteins in the cell types in which the protein is normally present and active. Functional proteins may be completely or partially absent, for example, due to transcriptional inactivation of the coding gene, or due to mutations in the coding gene that render the protein completely or partially nonfunctional. Examples of human diseases caused by the complete or partial inactivation of proteins include X-linked severe combined immunodeficiency (X-SCID) and adrenoleukodystrophy (X-ALD). X-SCID is caused by one or more mutations in the gene encoding a common gamma-chain protein, which is a component of the receptors for several interleukins involved in the development and maturation of B and T cells in the immune system. X-ALD is caused by one or more mutations in the gene for a peroxisome membrane transporter protein called ABCD1. Individuals with X-ALD have extremely high levels of long-chain fatty acids in their tissues throughout their body, which causes a variety of symptoms that can lead to mental disorders or death.

[0253] Attempts have been made to use gene therapy to treat several diseases caused by the deficiency or impairment of functional proteins in the cell types in which those proteins normally exist and are active. Gene therapy typically involves introducing a vector containing a gene encoding a functional form of the affected protein into the affected individual, and then expressing the functional protein to treat the disease. To date, success in gene therapy has been limited. Furthermore, specific embodiments of delivering mRNA using LNPs have been described, for example, in International Publications WO2018 / 006052 and WO2015 / 011633.

[0254] Therefore, there is a continuing need for improvements in expressing functional forms of proteins in humans suffering from diseases caused by the complete or partial deficiency of functional proteins, and for example, there is a need for improvements in nucleic acid (e.g., mRNA) delivery via methods and compositions that can further reduce the induction of an immune response to therapy. Certain embodiments of the present invention are useful in this context. Thus, in certain embodiments, polypeptide expression improves one or more symptoms of a disease or disorder. Certain compositions and methods of the present invention may be useful in treating human diseases caused by the deficiency or reduced levels of functional polypeptides in the human body. In other embodiments, certain compositions and methods of the present invention may be useful for the expression of vaccine antigens, for example, for treating cancer. Certain embodiments provide the use of lipid nanoparticles described herein as vaccines, for example, for use in delivering therapeutic mRNA to a target that needs it.

[0255] Self-amplifying RNA In certain embodiments, the nucleic acid is one or more self-amplified RNA molecules. Self-amplified RNA (sa-RNA) may also be referred to as self-replicating RNA, replicable RNA, replicon, or RepRNA. RepRNA, also referred to as self-amplified mRNA, is generated from a viral genome lacking at least one structural gene when derived from a positive-strand virus, and the RepRNA can be translated and replicated (and thus "self-amplified") without producing infectious progeny viruses. In certain embodiments, RepRNA technology may be used to insert a gene cassette encoding a desired target antigen. For example, an alphaviral genome is divided into two open reading frames (ORFs), where the first ORF encodes a protein of RNA-dependent RNA polymerase (replicase) and the second ORF encodes a structural protein. In a sa-RNA vaccine construct, the ORF encoding the viral structural protein may be replaced with any chosen antigen, but the viral replicase remains an essential part of the vaccine, facilitating intracellular amplification of RNA after immunization.

[0256] Preparation of lipid particles In certain embodiments, the present invention provides LNPs produced by a continuous mixing method, for example, a process comprising supplying an aqueous solution containing nucleic acid to a first reservoir, supplying an organic lipid solution to a second reservoir, and mixing the aqueous solution with the organic lipid solution such that the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce liposomes for encapsulating nucleic acid (e.g., interfering RNA or mRNA). The process and apparatus for carrying out the process are described in detail in U.S. Patent Publication No. 20040142025, which is incorporated herein by reference in its entirety for all purposes.

[0257] By continuously introducing lipids and a buffer solution into a mixing environment such as a mixing chamber, serial dilution of the lipid solution with the buffer solution occurs, thereby generating liposomes substantially instantaneously upon mixing. As used herein, the phrase (and variations thereof) "serially diluting a lipid solution with a buffer solution" generally means that the lipid solution is diluted sufficiently rapidly with a force sufficient to lead to vesicle formation in the hydration process. By mixing an aqueous solution containing nucleic acids with an organic lipid solution, the organic lipid solution undergoes serial dilution in the presence of a buffer solution (i.e., aqueous solution) to generate lipid nanoparticles.

[0258] LNPs formed using the continuous mixing method typically have sizes of approximately 40nm to 150nm, 40nm to 80nm, 40nm to 60nm, 50nm to 60nm, 50nm to 150nm, 60nm to 130nm, 70nm to 110nm, or 70nm to 90nm. The particles formed in this way do not aggregate and are optionally sized to obtain a uniform particle size.

[0259] In another embodiment, the present invention provides LNPs produced by a direct dilution process, which includes forming a liposome solution and immediately introducing the liposome solution directly into a collection container containing a controlled volume of dilution buffer. In a preferred embodiment, the collection container includes one or more components configured to agitate the contents of the collection container to facilitate dilution. In one embodiment, the amount of dilution buffer present in the collection container is substantially equal to the volume of liposome solution introduced therein. As a non-limiting example, a liposome solution in 45% ethanol would favor smaller particles if introduced into a collection container containing an equal volume of dilution buffer.

[0260] In yet another embodiment, the present invention provides a LNP produced by a direct dilution process, in which a third reservoir containing a dilution buffer is fluidly connected to a second mixing region. In this embodiment, the liposome solution formed in the first mixing region is immediately and directly mixed with the dilution buffer in the second mixing region. In a preferred embodiment, the second mixing region includes a T-connector arranged so that the flows of the liposome solution and the dilution buffer merge as 180° opposing flows, although connectors that result in a shallower angle, e.g., about 27° to about 180°, can be used. A pump mechanism delivers a controllable buffer flow to the second mixing region. In one embodiment, the flow rate of the dilution buffer supplied to the second mixing region is controlled to be substantially equal to the flow rate of the liposome solution introduced therefrom the first mixing region. This embodiment advantageously allows for further control of the flow of the dilution buffer mixing with the liposome solution in the second mixing region, and therefore also the concentration of the liposome solution in the buffer throughout the second mixing process. By controlling the flow rate of such dilution buffer, it becomes advantageous to form small particles at reduced concentrations.

[0261] The apparatus for carrying out these processes and these direct dilution processes is described in detail in U.S. Patent Publication No. 20070042031, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0262] LNPs formed using the direct dilution process typically have sizes of approximately 40nm to 150nm, 40nm to 80nm, 40nm to 60nm, 50nm to 60nm, 50nm to 150nm, 60nm to 130nm, 70nm to 110nm, or 70nm to 90nm. The particles formed in this way do not aggregate and are optionally sized to obtain a uniform particle size.

[0263] If necessary, the lipid particles of the present invention (e.g., LNPs) can be sized by any method available for sizing liposomes. Sizing can be performed to obtain a desired size range and a relatively narrow particle size distribution.

[0264] Several techniques are available for sizing particles to a desired size. One sizing method used for liposomes and equally applicable to the particles of the present invention is described in U.S. Patent No. 4,737,323, the disclosure of which is incorporated herein by reference in its entirety for all purposes. The particle suspension is sonicated by either in-tank sonication or probe sonication to gradually reduce the size to particles smaller than about 50 nm. Homogenization is another method that relies on shear energy to fragment larger particles into smaller particles. In a typical homogenization procedure, the particles are recirculated through a standard emulsion homogenizer until a selected particle size, typically about 60–80 nm, is observed. In both methods, the particle size distribution can be monitored by conventional laser particle size identification or QELS.

[0265] Extrusion of particles through a porous polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing particle size to a relatively distinct size distribution. Typically, the suspension is circulated through the membrane one or more times until the desired particle size distribution is obtained. The particles can be successively extruded by passing them through membranes with smaller pores to progressively reduce their size.

[0266] In some embodiments, the nucleic acids in the LNPs are pre-enriched, for example, as described in U.S. Patent Application No. 09 / 744,103 (this disclosure is incorporated herein by reference in its entirety for all purposes).

[0267] In other embodiments, the method further comprises adding a non-lipid polycation useful for performing cell lipofection using the composition of the present invention. Examples of suitable non-lipid polycations include hexadimethrin bromide (marketed under the trade name POLYBRENE® by Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts of hexadimethrin. Other suitable polycations include, for example, salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine, polyallylamine, and polyethyleneimine. These salts are preferably added after the particles have been formed.

[0268] Administration of lipid particles The lipid particles (e.g., LNPs) of the present invention are useful for introducing nucleic acids into cells after they have been formed. Therefore, the present invention also provides a method for introducing nucleic acids (e.g., interfering RNA or mRNA) into cells. This method is carried out in vitro or in vivo by first forming particles as described above, and then contacting the particles with cells for a certain period of time sufficient for delivery of nucleic acids into cells to occur.

[0269] The lipid particles (e.g., LNPs) of the present invention can be adsorbed to virtually any cell type with which they are mixed or in contact. Once adsorbed, the particles may be endocytized by a portion of the cell, exchange lipids with the cell membrane, or fuse with the cell. The introduction or incorporation of the nucleic acid (e.g., nucleic acid) portion of the particle may occur via any one of these pathways. In particular, when fusion occurs, the particle membrane is incorporated into the cell membrane and the contents of the particle mix with the intracellular fluid.

[0270] The lipid particles (e.g., LNPs) of the present invention can be administered either alone or in a mixture with a pharmaceutically acceptable carrier (e.g., physiological saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practices. Generally, buffered physiological saline (e.g., 135-150 mM NaCl) will be used as a pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, etc., containing glycoproteins for enhancing the stability of albumin, lipoproteins, globulins, etc. Further suitable carriers are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). As used herein, “carrier” includes all solvents, dispersions, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption retarders, buffers, carrier solutions, suspensions, colloids, etc. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not cause allergic or similar adverse reactions when administered to humans.

[0271] Pharmaceutically acceptable carriers are generally added after the formation of lipid particles. Therefore, after particle formation, the particles can be diluted in a pharmaceutically acceptable carrier such as physiological buffered saline.

[0272] The concentration of particles in a pharmaceutical formulation can vary considerably, from less than approximately 0.05% by weight, typically around 2-5% by weight, to as high as 10-90% by weight, and is selected primarily based on fluid volume, viscosity, etc., according to the chosen specific administration method. For example, the concentration may be increased to reduce the fluid load associated with treatment. This may be particularly desirable in patients with congestive heart failure associated with atherosclerosis or severe hypertension. Alternatively, particles composed of irritating lipids may be diluted to a low concentration to reduce inflammation at the administration site.

[0273] The pharmaceutical composition of the present invention may be sterilized by conventional, well-known sterilization techniques. The aqueous solution may be packaged for use, or filtered under sterile conditions and freeze-dried, and the freeze-dried preparation may be mixed with the sterile aqueous solution before administration. The composition may contain pharmaceutically acceptable auxiliary substances such as pH adjusters, buffers, and osmotic pressure adjusters, which are necessary to approximate physiological conditions, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride. Furthermore, the particulate suspension may contain lipid protectants to protect lipids from damage by free radicals and lipid peroxidation during storage. Lipophilic free radical quenchers such as alpha-tocopherol and water-soluble iron-specific chelating agents such as ferrioxamine are preferred.

[0274] In vivo administration Systemic delivery for in vivo therapy, such as delivery of therapeutic nucleic acids to distal target cells via bodily systems including circulation, has been achieved using nucleic acid-lipid particles, such as those described in PCT Publications WO05 / 007196, WO05 / 121348, WO05 / 120152, and WO04 / 002453 (these disclosures are incorporated herein by reference in their entirety for any purpose). The present invention also provides fully encapsulated lipid particles that protect nucleic acids from nuclease degradation in serum, are non-immunogenic, small in size, and suitable for repeated dosing.

[0275] In the case of in vivo administration, administration may be by any method known in the art, such as injection, oral administration, inhalation (e.g., intranasal or intratracheal), transdermal application, or rectal administration. Administration may be in single doses or divided doses. The pharmaceutical composition may be administered parenterally, i.e., intra-articular, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the pharmaceutical composition is administered intravenously or intraperitoneally by bolus injection (see, for example, U.S. Patent No. 5,286,634). Intracellular nucleic acid delivery is also 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). Further other methods for administering lipid-based therapeutics are described, for example, in U.S. Patents 3,993,754, 4,145,410, 4,235,871, 4,224,179, 4,522,803, and 4,588,578. Lipid particles can be administered by direct injection at the disease site or by injection distal to the disease site (see, for example, Culver, HUMAN GENE THERAPY, Mary Ann Liebert, Inc., Publishers, New York, pp. 70-71 (1994)). The disclosures of the above references are incorporated herein by reference in their entirety for all purposes.

[0276] The compositions of the present invention can be prepared into aerosol formulations, either alone or in combination with other suitable components, for administration by inhalation (e.g., intranasal or intratracheal) (i.e., they can be “sprayed”) (see Brigham et al., Am.J.Sci., 298:278 (1989)). The aerosol formulations can be placed in a pressurized, acceptable propellant such as dichlorodifluoromethane, propane, or nitrogen.

[0277] In certain embodiments, pharmaceutical compositions may be delivered by intranasal spray, inhalation, and / or other aerosol delivery vehicles. Methods for direct delivery of nucleic acid compositions to the lungs by transnasal aerosol spray are described, for example, in U.S. Patents 5,756,353 and 5,804,212. Similarly, drug delivery using intranasal particulate resins and lysophosphatidyl-glycerol compounds (U.S. Patent 5,725,871) is also well known in the pharmaceutical field. Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene-supported matrix is ​​described in U.S. Patent 5,780,045. The aforementioned patent disclosures are incorporated herein by reference in their entirety for all purposes.

[0278] For example, formulations suitable for parenteral administration via intra-articular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In the embodiment of the present invention, the composition is preferably administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, intravesically, or intrathecally.

[0279] Generally, for intravenous administration, lipid particle formulations are prepared using a suitable pharmaceutical carrier. Many pharmaceutically acceptable carriers can be used in the compositions and methods of the present invention. Suitable formulations for use in the present invention can be found, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). Various aqueous carriers, such as water, buffered water, 0.4% saline, 0.3% glycine, etc., may be used, and these aqueous carriers may contain glycoproteins for stability enhancement of albumin, lipoproteins, globulins, etc. Generally, buffered saline (135-150 mM NaCl) is used as a pharmaceutically acceptable carrier, but other suitable carriers may also suffice. These compositions can be sterilized by conventional liposome sterilization techniques such as filtration. The compositions may contain pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, osmotic pressure adjusters, and wetting agents, which are necessary to approximate physiological conditions, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. These compositions can be sterilized using the techniques described above, or they can be produced under sterile conditions by alternative methods. The resulting aqueous solutions may be packaged for use, or filtered under sterile conditions and freeze-dried, and the freeze-dried preparation may be mixed with the sterile aqueous solution before administration.

[0280] For certain applications, the lipid particles disclosed herein can be delivered to an individual by oral administration. The particles can be incorporated into excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, pills, lozenges, elixirs, mouthwashes, suspensions, oral sprays, syrups, wafers, etc. (see, for example, U.S. Patents 5,641,515, 5,580,579, and 5,792,451; these disclosures are incorporated herein by reference in their entirety for any purpose). These oral dosage forms may also contain, namely, binders, gelatin; excipients, lubricants, and / or flavorings. If the unit dosage form is a capsule, the dosage form may contain a liquid carrier in addition to the substances described above. Various other substances may be present as coatings or otherwise to alter the physical form of the dosing unit. Naturally, any substance used in the preparation of any unit dosage form should be pharmaceutically pure and substantially non-toxic in the amounts used.

[0281] Typically, these oral formulations may contain at least about 0.1% or more lipid particles, although the percentage of particles may naturally vary, and may conveniently be about 1% or 2% to about 60% or 70% or more of the total weight or volume of the formulation. Inevitably, the amount of particles in each therapeutically useful composition may be prepared in such a way that a suitable dosage is obtained at any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations are conscientious of those skilled in the art preparing such pharmaceutical formulations, and therefore, various dosages and treatment regimens may be desirable.

[0282] Formulations suitable for oral administration may consist of the following: (a) a packaged therapeutic agent in an effective amount, such as nucleic acid (e.g., interfering RNA or mRNA), suspended in a liquid solution, such as water, saline solution, or a diluent such as PEG400; (b) capsules, sachets, or tablets containing a predetermined amount of the therapeutic agent, such as nucleic acid (e.g., interfering RNA or mRNA), as a liquid, solid, granule, or gelatin; (c) a suspension in a suitable liquid; and (d) a suitable emulsion. Tablet forms may contain lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and one or more other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and pharmaceutically suitable carriers. The lozenge form may include a fragrance tablet containing a therapeutic agent in an inert base such as gelatin and glycerin, or a sucrose and gum arabic emulsion, or a gel, which contains a therapeutic agent, a therapeutic agent, and a carrier known in the art in addition to the therapeutic agent.

[0283] In other use cases, lipid particles can be incorporated into a wide range of topical dosage forms. For example, suspensions containing nucleic acid-lipid particles such as LNPs can be formulated and administered as gels, oils, emulsions, topical creams, pastes, ointments, lotions, foams, mousses, and the like.

[0284] When preparing the lipid particle pharmaceutical preparation of the present invention, it is preferable to use a large quantity of purified particles to reduce or remove empty particles or particles on which therapeutic agents such as nucleic acids have associated with the outer surface.

[0285] The method of the present invention can be carried out in a variety of hosts. Preferred hosts include mammalian species such as primates (e.g., humans and chimpanzees and other non-human primates), dogs, cats, horses, cattle, sheep, goats, rodents (e.g., rats and mice), rabbits, and pigs.

[0286] The amount of particles administered depends on the ratio of therapeutic agent (e.g., nucleic acid) to lipid, the specific therapeutic agent (e.g., nucleic acid) used, the disease or disorder being treated, the patient's age, weight, and condition, and the clinician's judgment, but is generally about 0.01 to about 50 mg / kg body weight, preferably about 0.1 to about 5 mg / kg body weight, or about 10 per administration (e.g., injection). 8 ~10 10 It must be a particle.

[0287] In vitro administration For in vitro applications, therapeutic agents such as nucleic acids (e.g., interfering RNA or mRNA) can be delivered to any cells grown in culture, regardless of whether they are of plant or animal origin, vertebrate or invertebrate, or of any tissue or type. In preferred embodiments, the cells are animal cells, more preferably mammalian cells, and most preferably human cells.

[0288] When performed in vitro, contact between cells and lipid particles occurs in a biologically compatible medium. Particle concentrations vary widely depending on the specific application, but are generally between approximately 1 μmol and 10 mmol. Treatment of cells with lipid particles is typically carried out at physiological temperature (approximately 37°C) for a period of approximately 1 to 48 hours, preferably approximately 2 to 4 hours.

[0289] In a group of preferred embodiments, the lipid particle suspension is divided into approximately 10 3 ~about 10 5 Cells / ml, more preferably about 2 × 10⁶ 4 It is added to cells plated with 60-80% confluent, having a cell density of cells / ml. The concentration of the suspension added to the cells is preferably about 0.01-0.2 μg / ml, more preferably about 0.1 μg / ml.

[0290] The delivery efficiency of LNPs or other lipid particles of the present invention can be optimized using an Endosomal Release Parameter (ERP) assay. The ERP assay is described in detail in U.S. Patent Publication No. 20030077829, the disclosure of which is incorporated herein by reference in its entirety for all purposes. More specifically, the objective of the ERP assay is to determine the effects of the various cationic lipid and helper lipid components of LNPs based on their relative effects on endosomal membrane binding / incorporation or fusion with / destabilization of the endosomal membrane. This assay allows for the quantitative determination of how each component of LNPs or other lipid particles affects delivery efficiency, thereby enabling the optimization of LNPs or other lipid particles. Typically, the ERP assay measures the expression of a reporter protein (e.g., luciferase, β-galactosidase, green fluorescent protein (GFP), etc.), and in some cases, LNP formulations optimized for expression plasmids are also suitable for encapsulating interfering RNA or mRNA. In other cases, ERP assays can be adapted to measure the downregulation of transcription or translation of a target sequence in the presence or absence of interfering RNA (e.g., siRNA). In other cases, ERP assays can be adapted to measure the expression of a target protein in the presence or absence of mRNA. By comparing ERPs for each of various LNPs or other lipid particles, an optimized system can be easily determined, for example, the LNP or other lipid particle that is most readily taken up into cells.

[0291] Cells for the delivery of lipid particles The compositions and methods of the present invention are used to treat a wide variety of cell types in vivo and in vitro. Suitable cells include, for example, hematopoietic progenitor (stem) cells, fibroblasts, keratinocytes, hepatocytes, endothelial cells, skeletal muscle and smooth muscle cells, osteoblasts, nerve cells, quiescent lymphocytes, terminally differentiated cells, slow-cycle or noncycling primary cells, parenchymal cells, lymphoid cells, epithelial cells, and osteocytes. In one embodiment, one or more nucleic acids (e.g., interfering RNA (e.g., siRNA) or mRNA) are delivered to cancer cells such as lung cancer cells, colon cancer cells, rectal cancer cells, anal cancer cells, bile duct cancer cells, small intestine cancer cells, 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, kidney cancer cells, central nervous system cancer cells, glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteogenic sarcoma tumor cells, and hematological cancer cells.

[0292] In vivo delivery of lipid particles such as LNPs encapsulating one or more nucleic acid molecules (e.g., interfering RNA (e.g., siRNA) or mRNA) is suitable for targeting cells of any cell type. This method and composition can be used in cells of a wide variety of vertebrates, including mammals such as dogs, cats, horses, cattle, sheep, goats, rodents (e.g., mice, rats, and guinea pigs), rabbits, pigs, and primates (e.g., monkeys, chimpanzees, and humans).

[0293] To the extent required, cell tissue culture is well known in this art. For example, Freshney, Culture of Animal Cells, a Manual of Basic Technique, 3rd Ed., Wiley-Liss, New York (1994), Kuchler et al., Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977), and the references cited therein provide general guidelines for cell culture. Cultured cell systems are often in the form of monolayer cells, but cell suspensions are also used.

[0294] Detection of lipid particles In some embodiments, the lipid particles of the present invention (e.g., LNPs) are detectable in a subject at approximately 1, 2, 3, 4, 5, 6, 7, 8 hours or more. In other embodiments, the lipid particles of the present invention (e.g., LNPs) are detectable in a subject approximately 8, 12, 24, 48, 60, 72, or 96 hours after administration of the particles, or approximately 6, 8, 10, 12, 14, 16, 18, 19, 22, 24, 25, or 28 days after administration of the particles. The presence of the particles can be detected from cells, tissues, or other biological samples derived from the subject. The particles can be detected, for example, by direct detection of the particles, by detection of therapeutic nucleic acids such as interfering RNA (e.g., siRNA) sequences or mRNA sequences, by detection of a target sequence of interest (i.e., by detecting changes in the expression of the target sequence), or by a combination thereof.

[0295] Particle detection Lipid particles of the present invention, such as LNPs, can be detected using any method known in the art. For example, labels can be directly or indirectly coupled to components of lipid particles using methods well known in the art. A wide variety of labels can be used, selected according to the required sensitivity, ease of conjugation with lipid particle components, stability requirements, and available measurement means and disposable provision. Suitable labels include spectroscopic labels such as fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon Green®; rhodamine and Texas Red, derivatives such as tetrarhodamine isothiocyanate (TRITC), digoxigenin, biotin, phycoerythrin, AMCA, CyDye®, etc.); 3 H, 125 I, 35 S, 14 C, 32 P, 33 This includes, but is not limited to, radioactive labels such as P; enzymes such as horseradish peroxidase and alkaline phosphatase; and spectroscopic colorimetric labels such as colloidal gold or colored glass or plastic beads such as polystyrene, polypropylene, and latex. The labels can be detected using any means known in the art.

[0296] Nucleic acid detection Nucleic acids (e.g., interfering RNA or mRNA) are detected and quantified by any number of means well known to those skilled in the art as described herein. Detection of nucleic acids can be performed by well-known methods such as Southern blot analysis, Northern blot analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. Further biochemical analytical methods such as spectrophotography, radiography, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and hyperdiffusion chromatography may also be used.

[0297] The choice of nucleic acid hybridization method is not critical. Various nucleic acid hybridization methods are known to those skilled in the art. For example, common methods include sandwich assays and competitive or substitution assays. Hybridization techniques are generally described, for example, in “Nucleic Acid Hybridization, A Practical Approach,” Eds. Hames and Higgins, IRL Press (1985).

[0298] The sensitivity of hybridization assays can be improved through the use of nucleic acid amplification systems that increase the amount of target nucleic acid detected. In vitro amplification techniques are known that are suitable for amplifying sequences for use as molecular probes, or for generating nucleic acid fragments for subsequent subcloning.Examples of sufficient techniques to guide parties through such in vitro amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification, and other RNA polymerase-mediated techniques (e.g., NASBA®), are Sambrook et al., In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2000), and Ausubel et al., SHORT PROTOCOLS IN MOLECULAR BIOLOGY, eds., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (2002), as well as U.S. Patent No. 4,683,202, PCR Protocols, A Guide to Methods and Applications (Innis et al. eds.), Academic Press Inc., San Diego, Calif. (1990), Arnheim & Levinson (Oct. 1, 1990), C&EN 36; The Journal of NIH Research,3:81(1991), Kwoh et al.,Proc.Natl.Acad.Sci.USA,86:1173(1989),Guatelli et al.,Proc.Natl.Acad.Sci.USA,87:1874(1990),Lomell et al. al., J. Clin. Chem., 35:1826 (1989), Landegren et al., Science, 241: 1077 (1988), Van Brunt, Biotechnology, 8: 291 (1990), Wu and Wallace, Gene, 4: 560 (1989), Barringer et al. al., Gene, 89:117 (1990), and Sooknanan and Malek, Biotechnology, 13:563 (1995). An improved method for cloning amplified nucleic acids in vitro is described in U.S. Patent No. 5,426,039.Other methods described in the art include nucleic acid sequence-based amplification (NASBA®, Cangene, Mississauga, Ontario) and Qβ-replicase systems. These systems can be used to directly identify variants if they are designed so that PCR or LCR primers extend or ligate only if the selected sequence is present. Alternatively, the selected sequence can be amplified, generally, using, for example, non-specific PCR primers, and then the amplified target region can be searched for for specific sequences exhibiting mutations. The disclosures of the references mentioned above are incorporated herein by reference in their entirety for all purposes.

[0299] For example, nucleic acids for use as probes in in vitro amplification methods, for use as gene probes, or as inhibitory components are typically chemically synthesized using an automated synthesizer, for example, Needham VanDevanter et al., Nucleic Acids Res., 12:6159 (1984), following the solid-phase phosphoramidite triester method described by Beaucage et al., Tetrahedron Letts., 22:1859 1862 (1981). If necessary, the purification of polynucleotides is usually carried out by either undenatured acrylamide gel electrophoresis or anion exchange HPLC, as described by Pearson et al., J. Chrom., 255:137 149 (1983). The sequences of synthetic polynucleotides can be verified using the chemical decomposition method described in Maxam and Gilbert (1980) in Grossman and Moldave (eds.), Academic Press, New York, Methods in Enzymology, 65:499.

[0300] An alternative method for measuring transcription levels is in situ hybridization. In situ hybridization assays are well-known and generally described in Angerer et al., Methods Enzymol., 152:649 (1987). In an in situ hybridization assay, cells are immobilized on a solid support, usually a glass slide. If DNA is to be searched, the cells are denatured with heat or alkali. The cells are then brought into contact with a hybridization solution at a moderate temperature to anneal the labeled specific probe. The probe is preferably labeled with a radioisotope or a fluorescent reporter. [Examples]

[0301] PEG2000-C-DMA (US8,936,942) is used in lipid nanoparticle (LNP) formulations that are in human clinical trials for a variety of applications, including oncology, vaccines, antivirals, and metabolic diseases. These LNP formulations are used to deliver therapeutic payloads containing, but not limited to, nucleic acids such as plasmid DNA, siRNA, mRNA, and self-replicating RNA. PEG-2000-C-DMA is a closely related structural analog of PEG-C-DOMG, which is used in FDA-approved Onpattro.

[0302] The properties of the alkyl domains of PEG lipids (e.g., size, number, saturation level) can influence the biological performance of LNPs in vivo. These alkyl domains act as "anchors" enabling formulation and incorporation into LNPs, leading to surface presentation of the PEG domain. In addition to enabling the formulation of small, stable particles and masking these particles in vivo to evade innate immune responses, PEG lipids are also crucial for biological activity. After in vivo administration, PEG lipids diffuse from LNPs and, in the case of liver-targeted formulations, are simultaneously replaced by APOEs that promote hepatocyte uptake via LDL receptors. Consequently, the rate at which PEG lipids are removed from LNPs is directly related to the effectiveness of the alkyl domain anchor and is a critical property.

[0303] As part of a strategy to optimize and improve LNP formulations for nucleic acid delivery, we designed and synthesized a series of PEG lipids in which two alkyl chains of the same length are bonded to a single atom (either carbon or nitrogen). This is in contrast to the design motif in C-DMA and DOMG, where the alkyl domains are linked via a glycidol linker. Several chemical functional groups were designed and investigated to link the PEG domain to the dialkyl domain. These novel PEG lipids, overall, feature alkyl chains of the same length (e.g., C 14 When compared, it performed at or above the benchmark PEG-2000-C-DMA. Surprisingly, certain motifs (e.g., carbamates and urea) were found to produce more effective particles than other related compounds.

[0304] General synthesis of symmetrical dialkylamines, as exemplified by the synthesis of ditetradecylamine (5) [ka] a. Synthesis of 4-methyl-N,N-ditetradecylbenzenesulfonamide (3) K2CO3 (20.18 g, 146.0 mmol), p-toluenesulfonamide (1) (5.00 g, 29.2 mmol), and 1-bromotetradecane (2) (21.7 ml, 73.0 mmol) were heated under reflux in DMF (75 ml) for 20 hours. The reaction mixture was cooled to room temperature, diluted with Et2O (100 ml), and filtered. The filtrate was washed with H2O (50 ml) and back-extracted with aqueous Et2O (100 ml). The combined organic layers were dried over (Na2SO4), filtered, and concentrated under vacuum. The residue was purified by automated flash chromatography (DCM / hexane 40 / 60) to obtain N,N-ditetradecyl-p-toluenesulfonamide (3) (15.86 g, 96%).

[0305] b. Synthesis of ditetradecylamine (5) A solution of 4-methyl-N,N-ditetradecylbenzenesulfonamide (3) (10 g, 17.7 mmol) in THF (5 ml) was added at 0°C to a solution of lithium naphthalenide (prepared by stirring naphthalene (4) (11.36 g, 88.7 mmol) and lithium metal (0.92 g, 133.0 mmol) in THF (40 ml) at room temperature for 1 hour), and the reaction was stirred at room temperature for 1 hour. MeOH (4 ml), followed by H2O (50 ml), was added, and the mixture was extracted with ether (100 ml). The organic matter was dried (Na2SO4), filtered, and concentrated under vacuum. The residue was purified by automated flash chromatography (SiO2 / Hex(50:50)) to obtain ditetradecylamine (5) (4.85 g, 66.7%).

[0306] Example 1. General synthesis of dialkylcarbamate PEG lipids as exemplified by compound (9). [ka] a. Synthesis of PEG2000 p-nitrocarbonate (8) Methoxy PEG2000 OH (6) and DIPEA (0.8 ml, 4.6 mmol) were stirred in DCM (100 ml) at room temperature. Bis(4-nitrophenyl carbonate) (7) (1.4 g, 4.6 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was washed with NaHCO3 (3 × 150 ml) and brine (150 ml), dried, filtered, and concentrated under vacuum. The residue was purified by automated flash chromatography (0-15% MeOH / DCM) to obtain PEG2000 p-nitrocarbonate (8).

[0307] b. Synthesis of dialkylcarbamate PEG lipids (9) PEG2000 p-nitrocarbonate (8) (1 g, 0.42 mmol), DIPEA (0.15 ml, 0.85 mmol), and ditetradecylamine (5) (0.174 g, 0.42 mmol) were heated under reflux in DCE for 16 hours. The reaction mixture was cooled, washed with saturated NaHCO3 (5 × 75 ml), dried in (MgSO4), and concentrated under vacuum. The residue was purified by automated flash chromatography (0-15% MeOH / DCM). This substance was further purified by tangential flow ultrafiltration (MWCO = 10000 kDA) with washing with 10 times the volume of water. The recovered aqueous solution was freeze-dried to obtain compound (9) (406 mg, 36%). 1 H NMR(400MHz,D6-DMSO)δ 4.21(m,3H),3.62(m,136H),3.38(s,3H),3.15(bs,5H),1.49(bs,4H),1.24(bs,35H),0.87(t,J=8Hz,6H).

[0308] Example 2. General synthesis of dialkylamide PEG lipids as exemplified by compound (13). [ka] a. Synthesis of 2-tetradecylhexadecanoic acid (11) Diethyl malonate (10) (10.0 g, 9.5 mmol) was added dropwise at 0°C to a suspension of NaH (60% dispersion in mineral oil) (2.75 g, 68.7 mmol) in anhydrous THF. 1-Bromotetradecane (2) (19.5 ml, 65.6 mmol) was added, and the reaction was refluxed for 18 hours. The reaction was cooled, quenched with MeOH, vacuum concentrated, and the residue was partitioned into hexane and H2O. The organic layer was washed with H2O and brine, dried (MgSO4), filtered, and vacuum concentrated. The residue was purified by automated flash chromatography (5% HCl / Hex). Subsequently, this material was subjected to a second alkylation step in the same manner as described above. The intermediate diethyl-2,2,dimyristalmalonate was dissolved in EtOH (250 ml), and KOH (24.5 g, 437 mmol) in H2O (250 ml) was added. The reaction mixture was stirred under reflux for 18 hours. The reaction mixture was cooled, poured onto ice, and acidified with 3 M HCl. The aqueous layer was extracted with SiO2, the organic matter was dried (MgSO4), and the mixture was concentrated under vacuum. The residue was heated at 170°C under vacuum without solvent until gas generation ceased. After cooling, the crude product was purified by automated flash chromatography (5% MeOH, DCM) to obtain 2-tetradecylhexadecanoic acid (11) (7.24 g, 24.4%).

[0309] b. Synthesis of dialkylamide PEG lipids (13) 2-Tetradecylhexadecanoic acid (11) (0.142 g, 0.31 mmol), methoxyPEG2000 amine (12) (500 mg, 0.25 mmol), HATU (0.143 g, 0.375 mmol), and DIPEA (0.13 mL, 0.75 mmol) were stirred in DCM at room temperature for 18 hours. The reaction mixture was diluted in DCM, sequentially washed with saturated NaHCO3 and brine, dried, filtered, and concentrated under vacuum. The residue was purified by automated flash chromatography (0-15% MeOH / DCM). The pure PEG-lipid was dissolved in water (8 mL) and lyophilized to obtain dialkylamide PEG-lipid (13) (365 mg, 60.0%). 1H NMR(400MHz,D6-DMSO)δ 3.81(m,1H),3.64(bs,179H),3.53(m,5H),3.45(m,1H),3.35(m,3H),2.8(s,6H),1.31(bs,53H),0.9(t,J=8Hz,6H).

[0310] Example 3. General procedure for the synthesis of dialkyl ester PEG lipids exemplified by compound (20) [ka] a. Synthesis of di-tert-butyl 2-tetradecylmalonate (15) NaH (60% dispersion in mineral oil) (453 mg, 11.3 mmol) was dissolved in DMF (100 mL) and cooled to 0°C. Di-tert-butylmalonate (14) (2.2 g, 10.3 mmol) was added and the reaction mixture was stirred for 10 minutes. 1-Bromotetradecane (2) (3.0 g, 10.8 mmol) was added and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was cooled to 0°C, quenched with saturated NH4Cl solution, and extracted with hexane. The organic layer was washed with brine, dried, filtered, and concentrated under vacuum. The residue was purified by automated flash chromatography (0-10% HCl / Hex) to obtain (15) (4.1 g, 95.7%).

[0311] b. Synthesis of di-tert-butyl 2,2-ditetradecylmalonate (16) A 60% NaH dispersion in mineral oil (433 mg, 10.8 mmol) was dissolved in DMF (100 mL) and cooled to 0°C. Di-tert-butyl 2-tetradecylmalonate (4.1 g, 9.9 mmol) was added, and the mixture was stirred for 10 minutes. 1-bromotetradecane (2) (2.87 g, 10.3 mmol) and NaI (0.44 g, 3.0 mmol) were added, and the reaction mixture was stirred at room temperature for 3 days. The reaction mixture was cooled to 0°C, quenched with saturated NH4Cl solution, and extracted with ethyl acetate. The organic layer was washed with brine, dried (MgSO4), filtered, and concentrated under vacuum. The residue was purified by automated flash chromatography (0-5% ethyl acetate / hexol) to obtain di-tert-butyl 2,2-ditetradecylmalonate (16) (5.0 g, 83.8%).

[0312] c. Synthesis of 2-tetradecylhexadecanoic acid (17) Di-tert-butyl 2,2-ditetradecylmalonate (16) (5.5 g, 9.1 mmol) was dissolved in DCM (40 mL), and TFA (20 mL) was added at 0°C. The reaction mixture was stirred at room temperature for 2 hours, and then azeotropically mixed twice with toluene. The residue was dissolved in xylene (30 mL) and stirred at 150°C for 18 hours. The reaction mixture was cooled to room temperature and concentrated under vacuum. The residue was dissolved in hexane (20 mL), heated to 50°C to dissolve, and heat-loaded onto a flash column. The product was purified by automated flash chromatography (0-30% Depositphotos / Hex) to obtain 2-tetradecylhexadecanoic acid (17) (2.2 g, 52.8%).

[0313] Synthesis of d.2-Tetradecylhexadecane-1-ol (18) A solution of 2-tetradecylhexadecanoic acid (17) (2.2 g, 5.0 mmol) in THF (100 mL) was added at 0°C to a stirred solution of LiAlH4 (364 mg, 9.6 mmol) in THF. The reaction mixture was warmed to room temperature and stirred for 18 hours. A saturated Na2SO4*10H2O solution was added dropwise while stirring at 0°C. The resulting PPT was removed by Celite filtration. The filtrate was concentrated under vacuum, and the residue was purified by automated flash chromatography (0-20% Â / hexane) to obtain 2-tetradecylhexadecan-1-ol (18) (1.9 g, 92.1%).

[0314] e. Synthesis of dialkyl ester PEG lipids (20) PEG2000-CO2H(19) (1.0 g, 0.45 mmol), 2-tetradecylhexadecane-1-ol(18) (0.3 g, 0.68 mmol), EDC.HCl (0.22 g, 1.1 mmol), DMAP (6 mg, 0.05 mmol), and DIPEA (0.18 g, 1.36 mmol) were dissolved in DCM (20 mL) at 0°C. The reaction mixture was warmed to room temperature and stirred for 3 days. Further EDC.HCl (0.22 g, 1.13 mmol) was added, and the reaction mixture was heated under reflux for 18 hours. After cooling, the reaction mixture was washed with brine, dried, filtered, and the filtrate was concentrated under vacuum. The residue was purified by automated flash chromatography (0-10% MeOH / DCM). The recovered product was further purified by tangential flow ultrafiltration (MWCO = 10,000 kDA) with washing with 10 times the volume of water. The recovered aqueous solution was freeze-dried to obtain dialkyl ester PEG lipid (20) (187 mg, 15.7%). 1H NMR (400 MHz, CDCl3) δ 4.14 (s, 2H), 4.05 (d, J = 4 Hz, 2H), 3.64 (PEG), 3.38 (S, 3H), 1.25 (bs, 54H), 0.88 (t, J = 8 Hz, 6H).

[0315] Example 4. General procedure for the synthesis of dialkyl ether PEG lipids exemplified by compound (24) [ka] a. Synthesis of nonacosan-15-ol (22) Myristylmagnesium bromide was prepared using standard Grignard conditions from 1-bromotetradecane(2) (20 g, 72.1 mmol) and magnesium shavings (1.75 g, 72.1 mmol) in anhydrous THF. After stirring at room temperature for 2 hours, ethyl formate(21) (5.8 ml, 72.1 mmol) was added dropwise, and the reaction mixture was stirred for 16 hours. After adding H2O (100 mL), 6 M HCl was slowly added until all magnesium was dissolved. The resulting PPT was removed by filtration, and the filtrate was concentrated under vacuum to obtain nonacosane-15-ol(22) (3.5 g, 22.8%), which was then used without further purification.

[0316] b. Synthesis of nonacosan-15-one (23) Nonacosane-15-ol (22) (500 mg, 1.2 mmol) was dissolved in DCM (20 mL). PCC (1.5 g, 3.5 mmol) on Si was added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered, and the filtrate was concentrated under vacuum to obtain crude nonacosane-15-one (23), which was used without purification.

[0317] c. Synthesis of dialkyl ether PEG lipids (24) Methoxy PEG2000-OH(6) (1.5 g, 0.75 mmol) was dissolved in anhydrous DCM (25 mL) and cooled to 0°C. Trimethylsilyl trifluoromethanesulfonate (0.22 g, 1 mmol) was added, and the reaction mixture was stirred for 20 minutes. Nonacosane-15-one(23) (211 mg, 0.5 mmol) and triethylsilane (116 mg, 1 mmol) were added sequentially. After 5 minutes, an additional trifluoromethanesulfonate (111 mg, 0.5 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours, quenched with 1 M NaOH (10 mL), and diluted with DCM (50 mL). The organic layer was washed with H2O (50 mL) and brine (50 mL), dried, and concentrated under vacuum in (MgSO4). The residue was purified by automated flash chromatography (0-15% MeOH / DCM) to obtain dialkyl ether PEG lipid (24) (635 mg, 54%). ¹H NMR (400 MHz, CDCl3) δ 3.8 (m, 2H), 3.63 (m, 156H), 3.37 (s, 3H), 3.22 (m, 2H), 1.24 (m, 48H), 0.87) t, J=8Hz, 6H).

[0318] Example 5. General procedure for the synthesis of dialkylurea PEG lipids exemplified by compound (26) [ka] a. Synthesis of dialkylurea PEG lipids (26) MethoxyPEG2000amine (12) (500 mg, 0.25 mmol) and DIPEA (0.096 mL, 0.55 mmol) were stirred in DCM (5 mL). Triphosgene (25) (27 mg, 0.093 mmol) was added, and the reaction mixture was stirred at room temperature for 4 hours. Ditetradecylamine (5) (102 mg, 0.25 mmol) in DCM (5 mL) was added dropwise, and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted in DCM, washed with brine, dried in (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by automated flash chromatography (0-15% MeOH / DCM). The recovered product was further purified by tangential flow ultrafiltration (10 washing volumes), lyophilized, and obtained dialkylurea PEG lipid (26) (250 mg, 0.102 mmol, 40.8%). 1H NMR(400MHz,CDCl3)δ 3.63(m,146H),3.54(m,6H),3.37(s,3H),3.15(t,J=8Hz,2H),1.82(bs,6H),1.51(bs,4H),1.24(m,40H),0.87(t,J=8Hz,6H).

[0319] General procedure for the synthesis of dialkylsulfonamide PEG lipids as illustrated by Example 6.(29) [ka] Synthesis of aN,N-ditetradecylethenesulfonamide (28) Ethenesulfonyl chloride (27) (154 mg, 1.2 mmol) was added to ditetradecylamine (5) (0.5 g, 1.2 mmol) and DIPEA (0.34 ml, 2.5 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 hours, concentrated under vacuum, and the residue was purified by automated flash chromatography (7% Âxane) to obtain N,N-ditetradecylethenesulfonamide (28) (190 mg, 31%). 1H NMR (400 MHz, CDCl3) δ 6.41 (m, 1H), 6.18 (D, J=16 Hz, 1H), (5.88, D, J=12 Hz, 1H), 3.09 (t, J=8 Hz, 4H), 1.56 (BS, 8H), 1.27 (BS, 41H), 0.87 (t, J=8 Hz, 6H).

[0320] b. Synthesis of dialkylsulfonamide PEG lipids (29) Methoxy PEG2000(6) (1.2 g, 0.56 mmol) and NaH (60% dispersion in mineral oil) (22 mg, 0.56 mmol) were stirred in anhydrous THF at room temperature for 30 minutes. N,N-ditetradecylethenesulfonamide(28) (280 mg, 0.56 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under vacuum, and the residue was purified by automated flash chromatography (7% MeOH / DCM) to obtain dialkylsulfonamide PEG lipid(29) (500 mg, 34%). 1H NMR(400MHz,CDCl3)δ 3.84(m,3H),3.58(m,110H),3.37(s,3H),3.21(t,J=8Hz,2H),3.13(t,t,8Hz,3H),1.72(BS,4H),1.56(BS,4H),1.27(BS,34H),0.87(t,J=8Hz,6H).

[0321] Example 7: Synthesis of Compound 30 [ka] Compound 30 was prepared according to Example 1, "General Synthesis of Dialkylcarbamate PEG Lipids." After purification, 465 mg of the desired product was obtained from suitable starting materials in 43% yield. 1H NMR (400 MHz, CDCl3) δ 4.20 (m, 2H), 3.80 (m, 1H), 3.66-3.45 (m, 154H), 3.37 (S, 3H), 3.17 (BS, 4H), 2.10 (BS, 5H), 1.48 (BS, 4H), 1.24 (BS, 36H), 0.87 (t, 6H, J=4Hz).

[0322] Example 8: Synthesis of Compound 31 [ka] Compound 31 was prepared according to the "General Synthesis of Dialkylcarbamate PEG Lipids" in Example 1. After purification, 664 mg of the required product was obtained in 58% yield from suitable starting materials. ¹H NMR (400 MHz, CDCl3) δ 4.20 (m, 2H), 3.80 (m, 1H), 3.66-3.45 (m, 167H), 3.37 (S, 3H), 3.17 (BS, 4H), 1.76 (BS, 5H), 1.48 (BS, 4H), 1.24 (BS, 50H), 0.87 (t, 6H, J=8Hz)

[0323] Example 9. Preparation of lipid nanoparticle formulation Lipid solutions were prepared using four components: PEG-bound lipid (either PEG-C-DMA control or the PEG lipid of the present invention), ionizable lipid ((6Z,16Z)-12-((Z)-deca-4-en-1-yl)docosa-6,16-dien-11-yl 5-(dimethylamino)pentanoic acid (described in US9,352,042)), cholesterol, and DSPC. The percentage molar ratios of the four components were 1.6:54.6:32.8:10.9, respectively, and the total lipid concentration in 100% ethanol of the solution was approximately 7 mg / mL. Firefly luciferase mRNA (TriLink Biotechnologies, L-7202) was diluted with acetate, pH 5 buffer, and nuclease-free water to obtain a 100 mM acetate solution (pH 5) of mRNA at the target concentration of 0.366 mg / mL. Equivolutes of lipid solution and nucleic acid solution were mixed at a flow rate of 400 mL / min using a T-connector and diluted to approximately four times the volume in pH 7.4 PBS. The formulation was placed in a Slide-A-Lyzer dialysis unit (MWCO 10,000) and dialyzed overnight with 10 mM Tris, 500 mM NaCl, and pH 8 buffer. After dialyzation, the formulation was concentrated to approximately 0.6 mg / mL using a VivaSpin concentrator (MWCO 100,000) and dialyzed overnight with 5 mM Tris, 10% sucrose, and pH 8 buffer. The formulation was filtered through a 0.2 μm syringe filter (PES membrane). Nucleic acid concentration was determined by the RiboGreen assay. Particle size and polydispersity were determined using a Malvern Nano Series Zetasizer.

[0324] Example 10. In vivo administration of LNP LNP preparations containing firefly luciferase mRNA were intravenously injected at a dose of 0.5 mg / kg into female BALB / c mice (6-8 weeks old). On the day of injection, the LNP stock was filtered and diluted to the required dose concentration with phosphate-buffered saline. Six hours after administration, the animals were euthanized with a lethal dose of ketamine / xylazine. Blood samples were collected in EDTA microtainer tubes and centrifuged at 16,000xg for 5 minutes at 4°C. Plasma samples were removed and stored at -80°C until cytokine analysis was performed. Liver samples (left lobe) were collected, weighed, and rapidly frozen in liquid nitrogen. Liver samples were stored in FastPrep® tubes at -80°C until analysis for luciferase activity was performed.

[0325] Example 11. Luciferase activity analysis Frozen liver aliquots were thawed and homogenized in 1 mL of 1x CCLR (cell culture lysis reagent) using a FastPrep® homogenizer. The homogenate was then centrifuged at 16,000 RPM for 10 minutes at 4°C. 20 μL of the supernatant was loaded into a 96-well white plate, and luminescence was measured after the addition of luciferase reagent (Promega Luciferase Assay System). Luciferase activity was determined by comparing the luminescence of the homogenized sample with that of a luciferase protein standard. To account for the quenching of luminescence by components in the liver homogenate, luciferase was added to the liver homogenate of untreated animals, and the resulting luminescence was measured. The obtained quenching factor was applied to all samples to obtain corrected luciferase activity, which was then normalized to per unit mass of the analyzed tissue.

[0326] The luciferase activity of 0.5 mg / kg LNP containing firefly luciferase mRNA and various PEG conjugate lipids was measured 6 hours after intravenous administration in BALB / c mice (n=4); the results are shown in the table below. [Table 1]

[0327] Example 12. Efficacy and tolerability of repeated administration in a rat model. Preparation of lipid nanoparticle formulations. These were prepared as described in Example 9, with the exception of the formulation containing human erythropoietin (EPO) mRNA (TriLink Biotechnologies, L-7209).

[0328] In vivo administration of LNP. A LNP preparation containing human EPO mRNA was intravenously injected at a dose of 0.25 mg / kg into male Sprague Dawley rats (7-8 weeks old). The animals received a total of three doses weekly (Q7D) on days 0, 7, and 14. On each injection day, the LNP stock was filtered and diluted with phosphate-buffered saline to the required dose concentration. Blood samples were collected in EDTA microtainer tubes before administration of the test substance, 6 hours after administration, and at the final time point (6 hours after the third administration), and centrifuged at 16,000xg for 5 minutes at 4°C. All plasma samples were stored at -80°C until EPO expression and anti-PEG antibody analysis were performed.

[0329] Anti-PEG antibody analysis. The presence of anti-PEG IgM and IgG antibodies in rat plasma samples was measured using ELISA. Millipore Multiscreen-HTS IP plates (Millipore, MSIPN4510) were coated with 0.5 mg / mL PEG lipid and kept at 37°C until dry. After blocking with 10% FBS in PBS and washing once with 1% FBS in PBS, the plasma samples were plated at a 1 / 30 dilution onto two separate plates. After three washes, goat anti-rat IgG conjugated with peroxidase (Cedarlane, 112-036-072) was added to one plate, and goat anti-rat IgM conjugated with peroxidase (Cedarlane, 112-036-075) was added to the other plate. After incubation and washing, TMB substrate (BD Biosciences, 555214) was added for color development, and the reaction was stopped with 2N sulfuric acid. For absorbance readings at 450 / 570 OD, the contents from each well were transferred to a transparent 96-well plate.

[0330] Nucleic acid payloads containing LNPs can be immunogenic if they circulate in the blood for extended periods. Antibodies (IgG / IgM) that recognize the surface chemistry of LNPs (typically PEG polymers) are produced and readily detectable in the blood. This can be problematic for repeated administration of LNPs, as anti-PEG antibodies induced by the initial dose detect and remove subsequent doses, leading to loss of efficacy and hypersensitivity reactions. This effect is exacerbated by more inflammatory payloads containing mRNA, which cannot be chemically modified to the same extent as other nucleic acid payloads (e.g., siRNA) as mitigation strategies. PEG lipids that promote faster hepatic uptake and more rapid clearance from the blood have been identified. PEG-C-DMA has been used as a benchmark and is used in several clinical siRNA-LNP products. Compounds of the present invention, such as compound 26, showed a much lower antibody (IgG / IgM) response than the benchmark PEG-C-DMA (see Figures 1 and 2).

[0331] EPO expression analysis. Human EPO concentrations in rat plasma were determined using an EPO ELISA assay (catalog number DEP00; R&D systems) according to the manufacturer's instructions.

[0332] EPO expression was measured 6 hours after intravenous administration of 0.25 mg / kg in SD rats (n=4). Loss of potency (human EPO expression) was observed with repeated administration of LNPs containing PEG-C-DMA, but not with compound 26 (see Figure 3). These data correlated with the level of anti-PEG antibodies induced by the initial administration, which recognize and rapidly eliminate subsequent administrations, rendering them ineffective.

[0333] Example 13. Single-dose OTC expression in a non-human primate model Preparation of lipid nanoparticle formulations. These were prepared as described in Example 9, with the exception of formulations containing mRNA encoding human ornithine transcarbamylase (OTC) custom synthesized at TriLink Biotechnologies.

[0334] In vivo administration of LNP. LNP preparations encapsulating human OTC mRNA were administered to naive cynomolgus monkeys (n=4; 2-3 years old) by intravenous infusion at a dose of 0.5 mg / kg over 60 minutes. On the day of administration, the LNP stock was filtered and diluted to the required dose concentration with 0.9% saline. Blood samples were collected in EDTA microtainer tubes at various points after the start of infusion and centrifuged at 16,000 × g for 5 minutes at 4°C. All plasma samples were stored at -80°C until lipid analysis was performed. At the end of the study (24 hours after administration), 500 mg of liver was collected from the left lobe and rapidly frozen in liquid nitrogen. Liver samples were stored in a freezer set to maintain -80°C until analysis for human OTC expression was performed by LC-MS.

[0335] Lipid clearance. The amount of ionizable lipids present in NHP plasma samples was measured over time using an LC-MS-based method (see Figure 4). Both PEG-C-DMA and compound 9 contain two C14 alkyl chains conjugated to a PEG polymer component with a molecular weight of approximately 2000 g / mol; however, compound 9 showed much faster clearance in NHP. This is consistent with the observation that the PEG-lipid-containing LNP of the present invention is less immunogenic than PEG-C-DMA. Compound 31 is an analogue of compound 9, but has a larger lipid anchor that retains it in the LNP (two C16 alkyl chains instead of a C14 chain). This suggests that the PEG-lipid with the larger anchor binds more strongly to the LNP, promoting a longer circulation time.

[0336] OTC protein expression. The amount of human OTC protein present in NHP liver samples 24 hours after administration was measured using an LC-MS-based method (see Figure 5). As expected, the slower pharmacokinetics of compound 31 resulted in lower protein expression in NHP liver, while both PEG-C-DMA and compound 9 showed similar levels of human OTC protein.

[0337] Example 14. Immunogenicity in a mouse OVA model after intramuscular administration. Preparation of lipid nanoparticle formulations. These were prepared as described in Example 9, with the exception of formulations containing ovalbumin (OVA) mRNA (TriLink Biotechnologies, L-7210).

[0338] In vivo administration of LNP. LNP preparations containing OVA mRNA were intramuscularly administered at a dose of 1 μg to BALB / c mice (7-8 weeks old) on day 0 (prime) and day 21 (boost). Blood samples were collected in EDTA microtainer tubes before administration and on days 7, 14, 21, and 28. End-stage blood was collected on day 35. Plasma samples were stored in a freezer set to maintain -80°C until analysis for anti-OVA IgG was performed by ELISA.

[0339] Anti-OVA IgG analysis. The production of anti-OVA IgG antibodies, a measure of efficacy in the vaccine setting, was measured using ELISA. (See Figure 6) Two PEG lipids of the present invention (compound 26, compound 30) were formulated into 1.5:50:38.5:10 (PEG-lipid:ionizable lipid:cholesterol:DSPC) and the benchmark compound PEG-DMG. PEG-DMG is a commercially available PEG lipid containing two C14 alkyl chains and is used in the Moderna COVID vaccine mRNA-1273, which was used as a benchmark in this study. Furthermore, the LNP compositions were compared with the LNP compositions of mRNA-1273 and the BNT162b2 COVID vaccine. After two IM administrations, LNPs containing either compound 26 or compound 30 showed similar anti-OVA IgG levels to the benchmark control (PEG-DMG, mRNA-1273, and BNT162b2).

[0340] The present invention will be described in more detail by specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present invention in any way.

[0341] It should be understood that the foregoing description is intended to be illustrative and not limiting. Many embodiments will become apparent to those skilled in the art upon reading the foregoing description. Therefore, the scope of the invention should not be determined by reference to the foregoing description, but rather by reference to the appended claims, along with the entire scope of the equivalents to which such claims are entitled. All disclosures of papers and references, including patent applications, patents, PCT publications, and Genbank accession numbers, are incorporated herein by reference for all purposes. Finally, preferred embodiments of the present invention are described in separate sections. [Embodiment 1] Compound of formula (I): [ka] or its salt (in the formula, R 1is H, (C1-C6) alkyl, or (C1-C6) alkanoyl, n is an integer in the range of approximately 10 to approximately 150, and L does not exist, and X is -C(=O)NR 2 R 3 is; or L is (C1-C6) alkyl, and X is -N(R 4 )C(=O)CH(R 2 )(R 3 ), -OCH(R 2 )(R 3 ), -C(=O)OCH2CH(R 2 )(R 3 ), -N(R 4 )C(=O)N(R 2 )(R 3 ), and -SO2N(R 2 )(R 3 Selected from the group consisting of; R 2 is (C 10 -C 20 ) is alkyl; R 3 is (C 10 -C 20 ) is alkyl; and R 4 (is H or (C1-C6) alkyl). [Embodiment 2] L does not exist, and X is -C(=O)NR 2 R 3 The compound described in Embodiment 1. [Embodiment 3] L is (C1-C6)alkyl and X is -N(R4)C(=O)CH(R 2 )(R 3 The compound described in Embodiment 1, which is the compound described in Embodiment 1. [Embodiment 4] L is (C1-C6) alkyl and X is -OCH(R 2 )(R 3 The compound described in Embodiment 1, which is the compound described in Embodiment 1. [Embodiment 5] L is (C1-C6) alkyl and X is -C(=O)OCH2CH(R) 2 )(R3 The compound described in Embodiment 1, which is the compound described in Embodiment 1. [Embodiment 6] L is (C1-C6) alkyl and X is -N(R 4 )C(=O)N(R 2 )(R 3 The compound described in Embodiment 1, which is the compound described in Embodiment 1. [Embodiment 7] L is (C1-C6) alkyl and X is -SO2N(R 2 )(R 3 The compound described in Embodiment 1, which is the compound described in Embodiment 1. [Embodiment 8] R 1 A compound according to any one of embodiments 1 to 7, wherein is H. [Embodiment 9] R 1 A compound according to any one of embodiments 1 to 7, wherein (C1-C6) alkyl. [Embodiment 10] R 1 A compound according to any one of embodiments 1 to 7, wherein the compound is methyl. [Embodiment 11] R 1 A compound according to any one of embodiments 1 to 7, wherein (C1-C6) alkanoyl. [Embodiment 12] R 1 A compound according to any one of Embodiments 1 to 7, wherein the parent compound is CH3C(=O)-. [Embodiment 13] A compound according to any of Embodiments 1 to 12, wherein n is an integer in the range of approximately 20 to approximately 100. [Embodiment 14] A compound according to any of Embodiments 1 to 12, wherein n is an integer in the range of approximately 20 to approximately 70. [Embodiment 15] A compound according to any of Embodiments 1 to 12, wherein n is an integer in the range of approximately 20 to approximately 60. [Embodiment 16] A compound according to any of Embodiments 1 to 12, wherein n is an integer in the range of approximately 20 to approximately 50. [Embodiment 17] A compound according to any of Embodiments 1 to 12, wherein n is an integer in the range of approximately 30 to approximately 100. [Embodiment 18] A compound according to any of Embodiments 1 to 12, wherein n is an integer in the range of approximately 40 to approximately 100. [Embodiment 19] A compound according to any of Embodiments 1 to 12, wherein n is an integer in the range of approximately 40 to approximately 60. [Embodiment 20] A compound according to any of Embodiments 1 to 12, wherein n is an integer in the range of approximately 40 to approximately 50. [Embodiment 21] A compound according to any of Embodiments 1 to 12, wherein n is an integer in the range of approximately 44 to approximately 46. [Embodiment 22] R 2 However, C 11 -alkyl, C 12 -alkyl, C 13 -alkyl, C 14 -alkyl, C 15 -alkyl, C 16 -alkyl, C 17 -alkyl, C 18 -alkyl, C 19 -alkyl or C 20 - A compound according to any one of embodiments 1 to 21, which is alkyl. [Embodiment 23] R 2 However, C 12 -alkyl, C 13 -alkyl, C 14 -alkyl, C 15 -alkyl, C 16 -alkyl, C 17 -alkyl or C 18 - A compound according to any one of embodiments 1 to 21, which is alkyl. [Embodiment 24] R 2 C 12 -alkyl, C 14 -alkyl or C 16 - A compound according to any one of embodiments 1 to 21, which is alkyl. [Embodiment 25] R2 C 14 - A compound according to any one of embodiments 1 to 21, which is alkyl. [Embodiment 26] R 3 However, C 11 -alkyl, C 12 -alkyl, C 13 -alkyl, C 14 -alkyl, C 15 -alkyl, C 16 -alkyl, C 17 -alkyl, C 18 -alkyl, C 19 -alkyl or C 20 - A compound according to any one of Embodiments 1 to 25, which is alkyl. [Embodiment 27] R 3 However, C 12 -alkyl, C 13 -alkyl, C 14 -alkyl, C 15 -alkyl, C 16 -alkyl, C 17 -alkyl or C 18 - A compound according to any one of Embodiments 1 to 25, which is alkyl. [Embodiment 28] R 3 C 12 -alkyl, C 14 -alkyl or C 16 - A compound according to any one of Embodiments 1 to 25, which is alkyl. [Embodiment 29] R 3 C 14 - A compound according to any one of Embodiments 1 to 25, which is alkyl. [Embodiment 30] R 4 A compound according to any one of embodiments 1 to 29, wherein is H. [Embodiment 31] R 4 A compound according to any one of embodiments 1 to 29, wherein is (C1-C6) alkyl. [Embodiment 32] R 4A compound according to any one of embodiments 1 to 29, wherein the compound is methyl. [Embodiment 33] [ka] Or a compound selected from salts thereof, as described in Embodiment 1. [Embodiment 34] A pharmaceutical composition comprising a compound or a salt thereof described in any of Embodiments 1 to 33 and a pharmaceutically acceptable carrier. [Embodiment 35] Lipid nanoparticles comprising a compound or a salt thereof as described in any of Embodiments 1 to 33. [Embodiment 36] Lipid nanoparticles according to Embodiment 35, further comprising cationic lipids, non-cationic lipids (e.g., phospholipids, e.g., DSPC), cholesterol, and a therapeutic agent. [Embodiment 37] The cationic lipids are: 3-(((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl)oxy)-N,N-dimethylpropan-1-amine; (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate; (6Z,16Z)-12-((Z)-deca-4-en-1-yl)docosa-6,16dien-11-yl 5-(dimethylamino)pentanoate (6Z,16Z)-12-((Z)-deca-4-en-1-yl)docosa-6,16-dien-11-yl6-(dimethylamino)hexanoate; N,N-dimethyl-4-(tris(((Z)-deca-4-en-1-yl)oxy)silyl)butan-1-amine; N,N-dimethyl-5-(tris(((Z)-deca-4-en-1-yl)oxy)silyl)pentan-1-amine; N,N-dimethyl-6-(tris(((Z)-deca-4-en-1-yl)oxy)silyl)he Xan-1-amine; 2-(methyl(4-(tris(((Z)-deca-4-en-1-yl)oxy)silyl)butyl)amino)ethane-1-ol; (6Z,16Z)-12-((6-(dimethylamino)hexanoyl)oxy)docosa-6,16-dien-11-yl(Z)-undeca-5-enoate; N1,N3-bis(4-(bis(((Z)-deca-4-en-1-yl)oxy)(methyl)silyl)butyl)-N1,N3-dimethylpropane-1,3-diamine; N1, N3-dimethyl-N1,N3-bis(4-(tris(((Z)-hepta-3-en-1-yl)oxy)silyl)butyl)propane-1,3-diamine; (1r,4r)-N1,N4-bis(4-(bis(((Z)-deca-4-en-1-yl)oxy)(methyl)silyl)butyl)-N1,N4-dimethylcyclohexane-1,4-diamine; 2,8-bis(4-(bis(((Z)-deca-4-en-1-yl)oxy)(methyl)silyl)butyl)-2,8-diazaspiro[4.5] Decane; or bis(2-butyloctyl)10-(N-(3-(dimethylamino)propyl)nonanamide)-nonadecanedioate; di(tridecane-7-yl)10-(N-(3-(dimethylamino)propyl)octanamide)-nonadecanedioate; di(tridecane-7-yl)10-(N-decyl-4-(dimethylamino)butanamide)nonadecanedioate; ((4-hydroxybutyl)azandiyl)bis(nonane-9,1-diyl (L)bis(2-butyloctanoate) or heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate; 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12dienoate or 3,6-bis(4-(bis(2-hydroxydodecyl) Amino)butyl)piperazine-2,5-dione; 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1-yl)amino)butyl)piperazine-2,5-dione; 1,1'-((2-(1-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperidine-4-yl)ethyl)azandiyl)bis(dodecane-2-ol); tetratridecyl 3,3 Lipid nanoparticles according to Embodiment 36, selected from ',3'',3'''-((azandiylbis(propane-3,1-diyl))bis(azantriyl))tetrapropionate or nonyl 8-((8,8-bis(octyloxy)octyl)(2-hydroxyethyl)amino)octanoate or di((Z)-nona-2-en-1-yl)8,8'-((((2-(dimethylamino)ethyl)thio)carbonyl)azandiyl)dioctanoate. [Embodiment 38] Lipid nanoparticles according to embodiment 36 or 37, wherein the therapeutic agent is a nucleic acid. [Embodiment 39] The lipid particle according to Embodiment 38, wherein the nucleic acid is DNA. [Embodiment 40] The lipid particle according to embodiment 38, wherein the nucleic acid is RNA. [Embodiment 41] Lipid particles according to Embodiment 39, wherein the DNA is in the form of an antisense molecule, plasmid DNA, pre-condensed DNA, PCR product, vector, expression cassette, chimeric sequence, or chromosomal DNA, or a combination thereof. [Embodiment 42] Lipid particles according to Embodiment 40, wherein the DNA is in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), or self-amplified RNA, or a combination thereof. [Embodiment 43] Lipid nanoparticles according to Embodiment 38, wherein the therapeutic agent is mRNA. [Embodiment 44] Lipid nanoparticles according to Embodiment 38, wherein the therapeutic agent is siRNA. [Embodiment 45] A pharmaceutical composition comprising lipid nanoparticles according to any of embodiments 35 to 44 and a pharmaceutically acceptable carrier. [Embodiment 46] A pharmaceutical composition according to Embodiment 45, formulated for intravenous or intramuscular administration. [Embodiment 47] A pharmaceutical composition according to Embodiment 46, formulated for intravenous administration. [Embodiment 48] A pharmaceutical composition according to Embodiment 46, formulated for intramuscular administration. [Embodiment 49] A method for delivering nucleic acids to cells, comprising contacting the cells with lipid nanoparticles described in any of embodiments 38 to 48. [Embodiment 50] A method for treating a disease characterized by a deficiency of a functional protein, comprising administering a lipid nanoparticle described in Embodiment 43 to a subject having the disease, wherein the mRNA encodes the functional protein or a protein having the same biological activity as the functional protein. [Embodiment 51] A method for treating a disease characterized by the overexpression of a polypeptide, comprising administering the lipid nanoparticles described in Embodiment 44 to a subject having the disease, wherein the siRNA targets the expression of the overexpressed polypeptide. [Embodiment 52] A method for delivering a vaccine to a target in need, comprising administering a therapeutically effective amount of lipid nanoparticles according to Embodiment 43 or a pharmaceutical composition according to Embodiment 48 to the target. [Embodiment 53] Lipid nanoparticles according to Embodiment 43 for therapeutic or prophylactic treatment of diseases characterized by functional protein deficiency. [Embodiment 54] Lipid nanoparticles according to Embodiment 44 for therapeutic or prophylactic treatment of diseases characterized by polypeptide overexpression. [Embodiment 55] Lipid nanoparticles according to embodiment 43 for use as a vaccine. [Embodiment 56] A pharmaceutical composition according to embodiment 48 for use as a vaccine.

Claims

1. Compounds of formula (I) or their salts: 【Chemistry 1】 [In the formula, R １ H, (C １ -C ６ ) alkyl, or (C １ -C ６ ) is alkanoyl, n is an integer in the range of approximately 10 to approximately 150, and L does not exist, and X is -C(=O)NR ２ R ３ is; or L is (C １ -C ６ ) alkyl, and X is selected from the group consisting of -N(R ４ )(C=O)N(R ２ )(R ３ ), and -SO ２ N(R ２ )(R ３ ); R ２ is (C １０ -C ２０ ) is alkyl; R ３ is (C １０ -C ２０ ) is alkyl; and R ４ is H or (C １ -C ６ ) It is alkyl.

2. L does not exist, and X is -C(=O)NR ２ R ３ The compound according to claim 1.

3. The compound according to claim 1, wherein L is (C1-C6)alkyl and X is -N(R4)C(=O)N(R2)(R3).

4. The compound according to claim 1, wherein L is (C1-C6) alkyl and X is -SO2N(R2)(R3).

5. R １ However, H, methyl, or CH ３ The compound according to any one of claims 1 to 4, wherein it is C(=O)-.

6. The compound according to any one of claims 1 to 5, wherein n is an integer in the range of approximately 20 to approximately 100.

7. The compound according to any one of claims 1 to 5, wherein n is an integer in the range of approximately 40 to approximately 60.

8. The compound according to any one of claims 1 to 5, wherein n is an integer in the range of approximately 44 to approximately 46.

9. R ２ However, C １１ - Alkyl, C １２ - Alkyl, C １３ - Alkyl, C １４ - Alkyl, C １５ - Alkyl, C １６ - Alkyl, C １７ - Alkyl, C １８ - Alkyl, C １９ - Alkyl or C ２０ - A compound according to any one of claims 1 to 8, wherein it is alkyl.

10. R ２ However, C １２ - Alkyl, C １４ - Alkyl or C １６ - A compound according to any one of claims 1 to 8, wherein it is alkyl.

11. R ２ C １４ - A compound according to any one of claims 1 to 8, wherein it is alkyl.

12. R ３ However, C １１ - Alkyl, C １２ - Alkyl, C １３ - Alkyl, C １４ - Alkyl, C １５ - Alkyl, C １６ - Alkyl, C １７ - Alkyl, C １８ - Alkyl, C １９ - Alkyl or C ２０ - The compound according to any one of claims 1 to 11, wherein it is alkyl.

13. R ３ However, C １２ - Alkyl, C １４ - Alkyl or C １６ - The compound according to any one of claims 1 to 11, wherein it is alkyl.

14. R ３ C １４ - The compound according to any one of claims 1 to 11, wherein it is alkyl.

15. R ４ The compound according to any one of claims 1 to 14, wherein the compound is H or methyl. 【Request Item 16】 【Chemistry 2】 The compound according to claim 1, or selected from salts thereof.

17. A pharmaceutical composition comprising a compound or salt thereof according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

18. Lipid nanoparticles comprising a compound or a salt thereof as described in any one of claims 1 to 16.

19. Lipid nanoparticles according to claim 18, further comprising a cationic lipid, a non-cationic lipid, cholesterol, and a therapeutic agent.

20. The cationic lipid is 3-(((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl)oxy)-N,N-dimethylpropane-1-amine; (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate; (6Z,16Z)-12-((Z)-deca-4-en-1-yl)docosa-6,16dien-11-yl5-(dimethylamino)pentanoate; (6Z,16Z)-12-((Z)-deca-4-en-1-yl)docosa-6,16-dien-11-yl6-(dimethylamino)hexanoate; N,N-dimethyl-4-(tris(((Z)-deca-4-en-1-yl)oxy)silyl)butan-1-amine; N,N-dimethyl-5-(tris(((Z)-deca-4-en-1-yl)oxy)silyl)pentan-1-amine; N,N-dimethyl-6-(tris(((Z)-deca-4-en-1-yl)oxy)silyl)hexane-1-amine; 2-(methyl(4-(tris(((Z)-deca-4-en-1-yl)oxy)silyl)butyl)amino)ethane-1-ol; (6Z,16Z)-12-((6-(dimethylamino)hexanoyl)oxy)docosa-6,16-dien-11-yl(Z)-unde-5-enoate; N1,N3-bis(4-(bis(((Z)-deca-4-en-1-yl)oxy)(methyl)silyl)butyl)-N1,N3-dimethylpropane-1,3-diamine; N1,N3-dimethyl-N1,N3-bis(4-(tris(((Z)-hepta-3-en-1-yl)oxy)silyl)butyl)propane-1,3-diamine; (1r,4r)-N1,N4-bis(4-(bis(( (Z)-deca-4-en-1-yl)oxy)(methyl)silyl)butyl)-N1,N4-dimethylcyclohexane-1,4-diamine; 2,8-bis(4-(bis(((Z)-deca-4-en-1-yl)oxy)(methyl)silyl)butyl)-2,8-diazaspiro[4.5]decane; or bis(2-butyloctyl)10-(N-(3-(dimethylamino)propyl)nonanamide)-nonadecanedioate; di(tridecane-7-yl)10-(N-(3-(dimethylamino)propyl)octanamide)-nonadecanedioate ;di(tridecane-7-yl)10-(N-decyl-4-(dimethylamino)butanamide)nonadecanedioate;((4-hydroxybutyl)azandiyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate) or heptadecan-9-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate;3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z, 12Z)-Octadeca-9,12-dienoate or 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione; 3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1-yl)amino)butyl)piperazine-2,5-dione; 1,1'-((2-(1-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperidine-4-yl)ethyl)azandiyl)bis(dodecane-2-ol);Lipid nanoparticles according to claim 19, selected from tetratridecyl 3,3',3'',3'''-((azandiylbis(propane-3,1-diyl))bis(azantriyl))tetrapropionate or nonyl 8-((8,8-bis(octyloxy)octyl)(2-hydroxyethyl)amino)octanoate or di((Z)-nona-2-en-1-yl)8,8'-((((2-(dimethylamino)ethyl)thio)carbonyl)azandiyl)dioctanoate;

21. The lipid nanoparticles according to claim 19 or 20, wherein the therapeutic agent is a nucleic acid.

22. The lipid nanoparticle according to claim 21, wherein the nucleic acid is DNA or RNA.

23. (i) The nucleic acid is DNA, and the DNA is in the form of an antisense molecule, plasmid DNA, pre-condensed DNA, PCR product, vector, expression cassette, chimeric sequence, or chromosomal DNA, or a combination thereof. (ii) The lipid nanoparticle according to claim 22, wherein the nucleic acid is RNA, and the RNA is in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), or self-amplified RNA, or a combination thereof.

24. (i) The therapeutic agent is mRNA, or (ii) The lipid nanoparticle according to claim 21, wherein the therapeutic agent is siRNA.

25. A pharmaceutical composition comprising lipid nanoparticles according to any one of claims 18 to 24 and a pharmaceutically acceptable carrier.

26. The pharmaceutical composition according to claim 25, formulated for intravenous or intramuscular administration.

27. A method for delivering nucleic acids to cells in vitro, comprising contacting the cells with lipid nanoparticles according to any one of claims 21 to 24.

28. A composition for treating a disease characterized by a deficiency of a functional protein, comprising the lipid nanoparticles described in claim 24, wherein the therapeutic agent is mRNA, and the mRNA encodes the functional protein or a protein having the same biological activity as the functional protein.

29. A composition for treating a disease characterized by the overexpression of a polypeptide, comprising the lipid nanoparticles described in claim 24, wherein the therapeutic agent is an siRNA, and the siRNA targets the expression of the overexpressed polypeptide.

30. A composition for delivering a vaccine to a target in need, comprising a therapeutically effective amount of lipid nanoparticles according to claim 24, wherein the therapeutic agent is mRNA.

31. (i) The therapeutic agent is mRNA and is intended for the therapeutic or prophylactic treatment of a disease characterized by a deficiency of a functional protein, or (ii) The therapeutic agent is an siRNA and is intended for the therapeutic or prophylactic treatment of a disease characterized by polypeptide overexpression, or (iii) The therapeutic agent is mRNA and is intended for use as a vaccine. Lipid nanoparticles according to claim 24.

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