Cationic lipids for the delivery of therapeutic lipid nanoparticles to hepatic stellate cells

Abstract

Certain embodiments of the present invention provide lipids useful for preparing lipid nanoparticles for delivering therapeutic agents, for example, to hepatic stellate cells.

Description

Cross-reference of related applications

[0001] This application claims priority to U.S. Patent Application No. 62 / 975,116, filed on 11 February 2020, which is incorporated herein by reference. [Background technology]

[0002] Nanoparticles containing cationic lipids have been used for the delivery of various therapeutic agents. Currently, the difficulties and costs associated with preparing the cationic lipid components of these nanoparticles limit their attractiveness for commercial development as delivery vehicles. Therefore, there is a need for more cationic lipids that can be incorporated into lipid nanoparticles. For example, there is a need for cationic lipids that can be prepared using cheaper and more efficient processes. There is also a need for relatively biodegradable cationic lipids so that the associated lipid nanoparticles degrade well after the delivery of the activator or therapeutic agent. Furthermore, there is a need for lipid nanoparticles with properties for delivering therapeutic agents to hepatic stellate cells, for example, for the treatment of hepatic fibrosis. [Overview of the Initiative] [Means for solving the problem]

[0003] The present invention provides cationic lipids that, when incorporated into lipid nanoparticles, are effective for delivering activators or therapeutic agents, such as nucleic acids, to hepatic stellate cells. These cationic lipids are also biodegradable so that the associated lipid nanoparticles degrade well after delivery of the activator or therapeutic agent.

[0004] Therefore, in one embodiment, the compound of formula (I) is provided herein: [ka] (In the formula, R 1 C2~C 30 It is hydrocarbyl; R 2 C2~C30 is a hydrocarbyl; R 3 is C1-C4 alkyl or -O-C2-C 30 is a hydrocarbyl; R 4 is C2-C 30 is a hydrocarbyl; R 5 is C2-C 30 is a hydrocarbyl; R 6 is C1-C4 alkyl or -O-C2-C 30 is a hydrocarbyl; X is a divalent linking group).

[0005] Also provided are lipid particles containing such compounds, and methods of using such lipid particles to specifically deliver, for example, a therapeutic agent to hepatic stellate cells to treat a disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] [Figure 1] Shows the leptin mRNA knockdown 48 hours after IV administration of LNP formulations in Balb / C mice (n = 4). LNPs using representative compounds of the present invention (shown in the 3rd and 4th columns) have the same potency as the reference compound (100; shown in the 2nd column) even though the dose is reduced to one-fourth (0.025 mg / kg instead of 0.1 mg / kg). [Figure 2] Represents the leptin mRNA knockdown 48 hours after IV administration of LNP formulations in Balb / C mice (n = 4). While the LNP using a representative compound of the present invention (shown in the 3rd column) has high specificity for HSCs, the LNP using a reference compound (101; shown in the 2nd column) designed for delivery to hepatocytes shows no knockdown in HSCs. This comparative data shows that the lipids of the present invention have the desired attributes that enable LNP delivery of therapeutic agents to HSCs. [Figure 3]This shows reelin mRNA knockdown 24 hours after IV administration of an LNP formulation in Balb / C mice (n=4). It demonstrates that LNP using the representative compound of the present invention achieves maximum knockdown in HSCs at approximately 0.03 mg / kg. [Figure 4] This shows reelin mRNA knockdown 24 hours after IV administration of the LNP formulation in Balb / C mice (n=4). The reference LNP (using compound 100; shown in columns 2-4) requires a higher dose (approximately 1 mg / kg) to achieve knockdown equivalent to that of the representative lipid of the present invention (shown in columns 5-7) (0.03 mg / kg). [Figure 5] A and B demonstrate the tolerability of LNPs formulated using representative lipids of the present invention (shown in columns 5-7), as they showed no significant increase in liver enzyme levels across a dose range with an upper limit of 3 mg / kg in this study. Group A also includes LNPs using compound 100 (shown in columns 2-4) as the first data set. [Figure 6] We demonstrate that LNPs formulated using minimally modified siRNA with representative lipids of the present invention exhibit a good duration of action (shown in the last 7-11 columns). Figure 6 also includes LNPs using compound 100, in columns 2-6 as the first data set. [Figure 7] We have demonstrated that LNPs formulated using the representative lipids of the present invention exhibit biodegradability, and that LNPs are rapidly removed from mouse liver. [Modes for carrying out the invention]

[0007] In one embodiment, the compound of formula (I) is provided herein: [ka] (In the formula, R 1 C2~C 30 It is hydrocarbyl; R2 C2~C 30 It is hydrocarbyl; R 3 These are C1-C4 alkyl groups, or -O-C2-C 30 It is hydrocarbyl; R 4 C2~C 30 It is hydrocarbyl; R 5 C2~C 30 It is hydrocarbyl; R 6 These are C1-C4 alkyl groups, or -O-C2-C 30 It is hydrocarbyl; X is a divalent linking group.

[0008] In one embodiment, R 1 is C2~C 20 It is hydrocarbil.

[0009] In one embodiment, R 1 is C2~C 15 It is hydrocarbil.

[0010] In one embodiment, R 1 is C2~C 10 It is hydrocarbil.

[0011] In one embodiment, R 1 is C5~C 20 It is hydrocarbil.

[0012] In one embodiment, R 1 (C2~C 20 ) Alkyl, (C2~C 20 ) Alkenyl, or (C2~C 20 ) This is Alkinnil.

[0013] In one embodiment, R 1 (C7~C 15 It is alkyl.

[0014] In one embodiment, R 1 (C7~C15 ) It is Alkenil.

[0015] In one embodiment, R 1 (C7~C 15 ) This is Alkinnil.

[0016] In one embodiment, R 1 It has only one double bond (C7~C 15 ) It is Alkenil.

[0017] In one embodiment, R 1 These are (Z)-4-decen-1-yl, 1-tridecyl, (Z)-3-hepta-1-yl, or (Z)-5-dodecen-1-yl.

[0018] In one embodiment, R 2 is C2~C 20 It is hydrocarbil.

[0019] In one embodiment, R 2 is C2~C 15 It is hydrocarbil.

[0020] In one embodiment, R 2 is C2~C 10 It is hydrocarbil.

[0021] In one embodiment, R 2 is C5~C 20 It is hydrocarbil.

[0022] In one embodiment, R 2 (C2~C 20 ) Alkyl, (C2~C 20 ) Alkenyl, or (C2~C 20 ) This is Alkinnil.

[0023] In one embodiment, R 2 (C7~C 15 It is alkyl.

[0024] In one embodiment, R 2(C7~C 15 ) It is Alkenil.

[0025] In one embodiment, R 2 (C7~C 15 ) This is Alkinnil.

[0026] In one embodiment, R 2 It has only one double bond (C7~C 15 ) It is Alkenil.

[0027] In one embodiment, R 2 These are (Z)-4-decen-1-yl, 1-tridecyl, (Z)-3-hepta-1-yl, or (Z)-5-dodecen-1-yl.

[0028] In one embodiment, R 3 These are C1-C4 alkyl groups.

[0029] In one embodiment, R 3 It is methyl.

[0030] In one embodiment, R 3 is -O-C2~C 20 It is hydrocarbil.

[0031] In one embodiment, R 3 is -O-C2~C 15 It is hydrocarbil.

[0032] In one embodiment, R 3 is -O-C2~C 10 It is hydrocarbil.

[0033] In one embodiment, R 3 is -O-C5~C 20 It is hydrocarbil.

[0034] In one embodiment, R 3 is -O-(C2~C 20 )alkyl, -O-(C2~C 20 ) Alkenyl, or -O-(C2~C20 ) is alkynyl.

[0035] In certain embodiments, R 3 is -O-(C7-C 15 ) alkyl.

[0036] In certain embodiments, R 3 is -O-(C7-C 15 ) alkenyl.

[0037] In certain embodiments, R 3 is -O-(C7-C 15 ) alkynyl.

[0038] In certain embodiments, R 3 is -O-(C7-C 15 ) alkenyl having only one double bond.

[0039] In certain embodiments, R 3 is (Z)-4-decen-1-yloxy, 1-tridecyloxy, (Z)-3-hepten-1-yloxy, or (Z)-5-dodecen-1-yloxy.

[0040] In certain embodiments, R 4 is C2-C 20 hydrocarbyl.

[0041] In certain embodiments, R 4 is C2-C 15 hydrocarbyl.

[0042] In certain embodiments, R 4 is C2-C 10 hydrocarbyl.

[0043] In certain embodiments, R 4 is C5-C 20 hydrocarbyl.

[0044] In certain embodiments, R 4 is, (C2-C 20) Alkyl, (C2~C 20 ) Alkenyl, or (C2~C 20 ) This is Alkinnil.

[0045] In one embodiment, R 4 (C7~C 15 It is alkyl.

[0046] In one embodiment, R 4 (C7~C 15 ) It is Alkenil.

[0047] In one embodiment, R 4 (C7~C 15 ) This is Alkinnil.

[0048] In one embodiment, R 4 It has only one double bond (C7~C 15 ) It is Alkenil.

[0049] In one embodiment, R 4 These are (Z)-4-decen-1-yl, 1-tridecyl, (Z)-3-hepta-1-yl, or (Z)-5-dodecen-1-yl.

[0050] In one embodiment, R 5 is C2~C 20 It is hydrocarbil.

[0051] In one embodiment, R 5 is C2~C 15 It is hydrocarbil.

[0052] In one embodiment, R 5 is C2~C 10 It is hydrocarbil.

[0053] In one embodiment, R 5 is C5~C 20 It is hydrocarbil.

[0054] In one embodiment, R 5 (C2~C20 ) Alkyl, (C2~C 20 ) Alkenyl, or (C2~C 20 ) This is Alkinnil.

[0055] In one embodiment, R 5 (C7~C 15 It is alkyl.

[0056] In one embodiment, R 5 (C7~C 15 ) It is Alkenil.

[0057] In one embodiment, R 5 (C7~C 15 ) This is Alkinnil.

[0058] In one embodiment, R 5 It has only one double bond (C7~C 15 ) It is Alkenil.

[0059] In one embodiment, R 5 These are (Z)-4-decen-1-yl, 1-tridecyl, (Z)-3-hepta-1-yl, or (Z)-5-dodecen-1-yl.

[0060] In one embodiment, R 6 These are C1-C4 alkyl groups.

[0061] In one embodiment, R 6 It is methyl.

[0062] In one embodiment, R 6 is -O-C2~C 20 It is hydrocarbil.

[0063] In one embodiment, R 6 is -O-C2~C 15 It is hydrocarbil.

[0064] In one embodiment, R 6 is -O-C2~C 10 It is hydrocarbil.

[0065] In one embodiment, R 6 is -O-C5~C 20 It is hydrocarbil.

[0066] In one embodiment, R 6 is -O-(C2~C 20 )alkyl, -O-(C2~C 20 ) Alkenyl, or -O-(C2~C 20 ) This is Alkinnil.

[0067] In one embodiment, R 6 is -O-(C7~C 15 It is alkyl.

[0068] In one embodiment, R 6 is -O-(C7~C 15 ) It is Alkenil.

[0069] In one embodiment, R 6 is -O-(C7~C 15 ) This is Alkinnil.

[0070] In one embodiment, R 6 -O-(C7~C) has only one double bond. 15 ) It is Alkenil.

[0071] In one embodiment, R 6 These are (Z)-4-decene-1-yloxy, 1-tridecyloxy, (Z)-3-hepta-1-yloxy, or (Z)-5-dodecene-1-yloxy.

[0072] In one embodiment, X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, where one or more of the carbon atoms (e.g., one, two, three, or four) are optionally (-O-) or (-NR a-), (divalent (C3-C8) cycloalkyl) or (divalent 3-12 member heterocycle), and the chain, the divalent (C3-C8) cycloalkyl, or the divalent 3-12 member heterocycle is optionally substituted with one or more substituents (e.g., one, two, three, or four) independently selected from the group consisting of (C1-C6) alkoxy, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, cyano, nitro, halo, hydroxy, oxo (=O), and carboxyl, and each R a The group is independently selected from H and (C1-C6) alkyl groups that are optionally substituted with one or more groups independently selected from halo, hydroxy, and (C1-C6) alkoxy groups.

[0073] In one embodiment, X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, where one or more of the carbon atoms (e.g., one, two, three, or four) are optionally (-O-) or (-NR a -), (divalent (C3-C8) cycloalkyl) or (divalent 3-8 membered heterocycle), and the chain, the divalent (C3-C8) cycloalkyl, or the divalent 3-8 membered heterocycle is optionally substituted with one or more substituents (e.g., one, two, three, or four) independently selected from the group consisting of (C1-C6) alkoxy, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, cyano, nitro, halo, hydroxy, oxo (=O), and carboxyl, and each R a These are independently selected from H and (C1-C6) alkyl groups.

[0074] In one embodiment, X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, where one or more of the carbon atoms (e.g., one, two, three, or four) are optionally (-NR a -) or (a divalent 3-12 member heterocycle), and each R aThe (C1-C6) alkyl group is independently selected from (C1-C6) alkyl groups that are optionally substituted with one or more groups independently selected from halo, hydroxy, and (C1-C6) alkoxy groups.

[0075] In one embodiment, X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, where one or more of the carbon atoms (e.g., one, two, three, or four) are optionally (-NR a -) or (a divalent 3- to 8-membered heterocycle), and each R a The (C1-C6) alkyl group is independently selected.

[0076] In one embodiment, X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, where one or more of the carbon atoms (e.g., one, two, three, or four) are (-NR a -) is replaced, and each R a The (C1-C6) alkyl group is independently selected from (C1-C6) alkyl groups that are optionally substituted with one or more groups independently selected from halo, hydroxy, and (C1-C6) alkoxy groups.

[0077] In one embodiment, X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, where one or more of the carbon atoms (e.g., one, two, three, or four) are (-NR a -) is replaced, and each R a The (C1-C6) alkyl group is independently selected.

[0078] In one embodiment, X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, where one or more of these carbon atoms (e.g., one, two, three, or four) are optionally replaced by a divalent 3- to 12-membered heterocycle.

[0079] In one embodiment, X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, where one or more of these carbon atoms (e.g., one, two, three, or four) are optionally replaced by a divalent 3- to 8-membered heterocycle.

[0080] In one embodiment, X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 6 to 12 carbon atoms, where one or more of the carbon atoms (e.g., one, two, three, or four) are optionally replaced by piperazine-1,4-diyl.

[0081] In one embodiment, X is [ka] [ka] (In the formula, a is 2, 3, 4, or 5; b is 2, 3, 4, or 5; c is 2, 3, 4, or 5; d is 2, 3, 4, or 5; e is 2, 3, 4, 5, 6, 7, or 8; f is 2, 3, 4, 5, 6, 7, or 8; Each R a This is independently selected from the group consisting of H and (C1-C6) alkyl groups which are optionally substituted with one or more groups independently selected from halo, hydroxy, and (C1-C6) alkoxy groups.

[0082] In one embodiment, X is [ka] (In the formula, a is 2, 3, 4, or 5; b is 2, 3, 4, or 5; c is 2, 3, 4, or 5; d is 2, 3, 4, or 5; e is 2, 3, 4, 5, 6, 7, or 8; f is 2, 3, 4, 5, 6, 7, or 8; Each R a The elements are independently selected from the group consisting of H and (C1-C6) alkyl groups.

[0083] In one embodiment, X is [ka] [ka] [ka] [ka] It is selected from the group consisting of the following.

[0084] In one embodiment, X is [ka] It is selected from the group consisting of the following.

[0085] In one embodiment, the compound is selected from the compounds described in the examples, for example, compounds 2 to 42, for example, compounds of any one formula from 2 to 30.

[0086] Further provided herein are lipid particles containing the compounds described herein.

[0087] In one embodiment, the lipid particles further comprise noncationic lipids.

[0088] In one embodiment, the lipid particles further include conjugate lipids that suppress particle aggregation.

[0089] In one embodiment, the lipid particles further comprise a therapeutic agent.

[0090] In one embodiment, the therapeutic agent is a nucleic acid therapeutic agent.

[0091] In one embodiment, the therapeutic agent is an interfering RNA agent.

[0092] In one embodiment, the therapeutic agent is siRNA.

[0093] In one embodiment, the therapeutic agent is mRNA.

[0094] In one embodiment, the nucleic acid therapeutic agent comprises at least one modified nucleotide.

[0095] In one embodiment, the nucleic acid comprises at least one 2'-O-methyl(2'OMe)nucleotide.

[0096] In one embodiment, the noncationic lipid is cholesterol or a derivative thereof.

[0097] In one embodiment, the noncationic lipid is cholesterol.

[0098] In one embodiment, the noncationic lipid includes phospholipids.

[0099] In one embodiment, the noncationic lipid includes a mixture of phospholipids and cholesterol.

[0100] In one embodiment, the phospholipid is distearoylphosphatidylcholine (DSPC).

[0101] In one embodiment, the conjugated lipid is a polyethylene glycol (PEG)-lipid conjugate.

[0102] In one embodiment, the PEG-lipid conjugate is a PEG-dimyristyloxypropyl (PEG-DMA) conjugate.

[0103] Further provided herein are compositions comprising the compounds or lipid particles described herein.

[0104] Further provided herein are pharmaceutical compositions comprising the compounds or lipid particles described herein and a pharmaceutically acceptable carrier.

[0105] Further provided is a method for in vivo delivery of a therapeutic agent, comprising administering lipid particles described herein to a mammalian subject.

[0106] Also provided are lipid particles described herein for use in the in vivo delivery of therapeutic agents to mammals.

[0107] Furthermore, the use of lipid particles described herein for preparing pharmaceuticals for in vivo delivery of therapeutic agents to mammals is also provided.

[0108] Further provided is a method for treating a disease or disorder in a mammalian subject requiring treatment of the disease or disorder, the method comprising administering a therapeutically effective amount of the lipid particles described herein to the mammalian subject.

[0109] In one embodiment, the disease or disorder is hepatic fibrosis.

[0110] In one embodiment, the disease or disorder is non-alcoholic steatohepatitis (NASH).

[0111] In one embodiment, the disease or disorder is alcoholic steatohepatitis (ASH).

[0112] In one embodiment, the disease or disorder is non-alcoholic steatohepatitis (NASH) or alcoholic steatohepatitis (ASH) associated with hepatic fibrosis.

[0113] Also provided is a method for delivering a therapeutic agent to hepatic stellate cells (HSCs) in vivo or in vitro, comprising contacting the HSCs with lipid particles described herein.

[0114] Hepatic fibrosis is caused by excessive accumulation of extracellular matrix during chronic liver injury. Activation of hepatic stellate cells (HSCs) is a crucial step during hepatic fibrosis. Targeted delivery of therapeutic agents to HSCs, such as activated HSCs, can be important for the successful treatment of hepatic fibrosis. Several protein markers have been found to be overexpressed in activated HSCs, and their ligands have been used to specifically deliver various antifibrotic agents (see, e.g., Chen et al., Journal of Pharmacology and Experimental Therapeutics, 2019, 370(3)695-702). However, therapeutic agent delivery using other systems, such as lipid nanoparticles (LNPs), is being sought as an alternative means of delivering therapeutic agents to HSCs.

[0115] Hepatic fibrosis is caused by the formation of an abnormally large amount of scar tissue in the liver. Hepatic fibrosis occurs when the liver attempts to repair and replace damaged cells. Various disorders and drugs can damage the liver and cause fibrosis.

[0116] Non-alcoholic fatty liver disease (NAFLD) is a condition characterized by the accumulation of triglycerides in the liver. Non-alcoholic steatohepatitis (NASH) is a type of NAFLD. NASH is associated with inflammatory changes and hepatocyte damage. NASH is a major cause of liver disease and often progresses to hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) have similar pathogenesis and histopathology, but differ in etiology and epidemiology. NASH and ASH are advanced stages of non-alcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (AFLD), respectively. Alcoholic steatohepatitis (ASH) is a chronic, progressive liver disease characterized by hepatic fibrosis and possible necrosis of liver tissue, caused by excessive and prolonged alcohol use. Women are more susceptible to this disease than men because they have a weaker alcohol metabolism.

[0117] Hepatic fibrosis is a significant underlying cause of liver dysfunction and is often fatal. Progression to cirrhosis and HCC leads to eventual liver failure, thus requiring a liver transplant. Currently, there are approximately 3.8 million patients with NASH-associated fibrosis (F2 and beyond) in the United States. Physicians typically recommend weight loss to treat NAFLD and NASH. While weight loss can reduce fat, inflammation, and fibrosis in the liver, there are no approved drugs to treat NAFLD and NASH. Specifically, there are no approved drugs to treat hepatic fibrosis (Clin Liver Dis. 2008 Nov;12(4):733-46, N Engl J Med. 2017 Nov 23;377(21):2063-2072, J Hepatol. 2017 Dec;67(6):1265-127). Therefore, new treatment options, including delivery options, are needed to treat hepatic fibrosis, for example, in association with NASH or ASH.

[0118] definition As used herein, the following terms have the meanings set forth below unless otherwise specified.

[0119] Halo or halogen is fluoro, chloro, bromo or iodo.

[0120] Alkyl, alkoxy, alkenyl, alkynyl, etc. represent both straight-chain and branched-chain groups, but reference to an individual radical, for example propyl, implies only the straight-chain radical, and branched-chain isomers, for example isopropyl, are specifically referred to.

[0121] The term "hydrocarbyl", unless otherwise specified, means, by itself or as part of another substituent, a straight-chain or branched-chain hydrocarbon radical having the specified number of carbon atoms (for example C 1-8 means 1 to 8 carbons) and optionally having one or more double bonds and / or one or more triple bonds. Thus, hydrocarbyl includes alkyl groups, alkenyl groups and alkynyl groups.

[0122] The term "alkyl", unless otherwise specified, means, by itself or as part of another substituent, a straight-chain or branched-chain hydrocarbon radical having the specified number of carbon atoms (for example C 1-20 means 1 to 20 carbons). Examples include (C1-C 20 )alkyl, (C2-C 20 )alkyl, (C5-C 20 )alkyl, (C5-C 15 )alkyl and (C 10 -C 20 )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 higher homologs and isomers.

[0123] The term "alkenyl" refers to an unsaturated alkyl radical having one or more double bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, clotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), and more advanced homologs and isomers.

[0124] The term "alkynyl" refers to an unsaturated alkyl radical having one or more triple bonds. Examples of such unsaturated alkyl groups include ethynyl, 1- and 3-propynyl, 3-butynyl, and their more advanced homologs and isomers.

[0125] The term "alkoxy" refers to an alkyl group that is bonded to the rest of a molecule via an oxygen atom ("oxy").

[0126] The term "cycloalkyl" refers to a saturated or partially unsaturated (non-aromatic) whole carbon ring (e.g., a (C3-C8) carbon ring) having 3 to 8 carbon atoms.

[0127] The term “heterocycle” refers to a saturated or partially unsaturated monocycle having at least one atom other than carbon, selected from the group consisting of oxygen, nitrogen, and sulfur. Therefore, the term includes saturated or partially unsaturated monocycles (e.g., 3, 4, 5, 6, 7, or 8-membered rings) having about 1 to 6 carbon atoms and about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. Sulfur and nitrogen atoms may be present in their oxidized forms. Exemplary heterocycles include, but are not limited to, azetidinyl, tetrahydrofuranyl, piperazinyl, and piperidinyl.

[0128] As used herein, the term "alkoxycarbonyl" refers to the group (alkyl)-OC(=O)-, and the term alkyl has the meaning defined herein.

[0129] As used herein, the term "alkanoyloxy" refers to the group (alkyl)-C(=O)-O-, and the term alkyl has the meaning defined herein.

[0130] As used herein, the term “heteroatom” is intended to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

[0131] Where used herein, a wavy line intersects a bond in the chemical structure. [ka] The tilde indicates the point in the chemical structure where a bond intersecting the wavy line is bonded to the rest of the molecule.

[0132] 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 (e.g., 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.

[0133] 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 approximately 15-30, 15-25, or 19-25 (double-stranded) nucleotides, preferably approximately 20-24, 21-22, or 21-23 (double-stranded) nucleotides (e.g., each complementary of a double-stranded siRNA). The sequences are 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides long, preferably about 20-24, 21-22, or 21-23 nucleotides long, and the double-stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs long, preferably about 18-22, 19-20, or 19-21 base pairs long). The siRNA double-stranded may contain a 3' overhang and a 5' phosphate terminus of about 1-4 nucleotides or about 2-3 nucleotides. Examples of siRNAs include, but are not limited to, double-stranded polynucleotide molecules assembled from two separate strand molecules (where one strand is a sense strand and the other is a complementary antisense strand); double-stranded polynucleotide molecules assembled from single-stranded molecules (where 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 a stem with two or more loop structures and self-complementary sense and antisense regions (where this cyclic polynucleotide can be processed in vivo or in vitro to produce an active double-stranded siRNA molecule).

[0134] Preferably, siRNA is chemically synthesized. siRNA can also be produced by cleaving longer dsRNA (e.g., dsRNA longer than approximately 25 nucleotides) with E. coli RNase III or a 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 longer. The dsRNA may encode an entire gene transcript or a portion of a gene transcript. In certain cases, siRNA may be encoded by a plasmid (for example, transcribed as a sequence that spontaneously folds into a double helix with a hairpin loop).

[0135] 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 does not have 100% complementarity to its target sequence. An interfering RNA may have at least one, two, three, four, five, six, or more mismatch regions. The 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.

[0136] An "effective dose" or "therapeutic effective dose" of an activator or therapeutic agent, such as a nucleic acid (e.g., interfering RNA or mRNA), is the amount sufficient to produce the desired effect, e.g., inhibition of the expression of a target sequence compared to the normal expression level detected in the absence of the 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 a protein normally expressed in a cell type in the body, and the therapeutically effective amount of mRNA is an amount that causes the production of at least 50% (e.g., at least 60%, or at least 70%, or at least 80%, or at least 90%) of the amount of encoded 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, protein or RNA-level testing 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.

[0137] 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 measured by comparing it 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 usually 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.

[0138] To “reduce,” “decrease,” 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 measured by comparing it 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 usually 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.

[0139] As used herein, the term “responder cell” refers to a cell, preferably a mammalian cell, that produces a detectable immune response upon contact with an immunostimulatory interfering RNA, such as unmodified siRNA. Exemplary responder cells include, for example, dendritic cells, macrophages, peripheral blood mononuclear cells (PBMCs), and spleen cells. Detectable immune responses include 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.

[0140] "Substantial identity" refers to a sequence that hybridizes to a reference sequence under stringent conditions, or a sequence that has a specific percentage of identity across a specific region of the reference sequence.

[0141] The term "stringent hybridization conditions" refers to conditions under which a nucleic acid, typically in a complex mixture of nucleic acids, hybridizes to its target sequence but not to other sequences. Stringent conditions are sequence-dependent and will vary in different situations. Longer sequences hybridize, in particular, at higher temperatures. Extensive guidance on nucleic acid hybridization can be 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 involve the thermal melting point (T) of a particular sequence at a given ionic strength pH. m It is selected to be approximately 5-10°C lower than ). mis the temperature at which 50% of the probe complementary to the target hybridizes to the target sequence at equilibrium (at a defined ionic strength, pH, and nucleic concentration), where the target sequence is present in excess, T m at which 50% of the probe is occupied at equilibrium. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal is at least 2-fold, preferably 10-fold, of the background hybridization.

[0142] Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5×SSC, and 1% SDS, incubated at 42 °C, or incubated at 5×SSC, 1% SDS, 65 °C, and washed in 0.2×SSC and 0.1% SDS at 65 °C. For PCR, low stringency amplification is usually at a temperature of about 36 °C, but the annealing temperature can vary from about 32 °C to 48 °C depending on the primer length. For high stringency PCR amplification, it is usually at a temperature of about 62 °C, but the high stringency annealing temperature can range from about 50 °C to about 65 °C depending on the primer length and specificity. Typical cycle conditions for both high stringency amplification and low stringency amplification include a denaturation step at 90 °C - 95 °C for 30 seconds - 2 minutes, an annealing step following for 30 seconds - 2 minutes, and an extension step at about 72 °C for 1 - 2 minutes. Protocols and guidelines for low stringency amplification reactions and high stringency amplification reactions are shown, for example, in Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y. (1990).

[0143] Nucleic acids that do not hybridize to one another under stringent conditions are still substantially identical if the polypeptides they encode are substantially the same. 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 in a buffer of 40% formamide, 1M NaCl, and 1% SDS at 37°C, and washing with 1×SSC at 45°C. Positive hybridization is at least twice the background. Those skilled in the art will readily understand that similar stringency conditions can be obtained by utilizing alternative hybridization and washing conditions. Further guidelines for determining hybridization parameters are provided in numerous references, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds.

[0144] The terms “substantially identical” or “substantially identical” in relation to two or more nucleic acids refer to two or more sequences or subsequences that, when compared and aligned to the greatest extent possible across a comparison window, or using one of the following sequence comparison algorithms, or across a designated region measured by manual alignment and visual inspection, are identical or have a specific percentage of identical nucleotides (e.g., at least about 60%, preferably at least about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity across a given region). This definition also refers to the complement of sequences, where the context indicates. Preferably, substantial identity exists over a region of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

[0145] For sequence comparison, typically one sequence acts as a reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into the computer, and subsequence coordinates are specified as needed, along with the sequence algorithm program parameters. Default program parameters may be used, or alternative parameters may 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.

[0146] 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)).

[0147] 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 in conjunction with the parameters described herein to determine the sequence identity percentage of nucleic acids according to the present invention. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0148] 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.

[0149] As used herein, the term “nucleic acid” refers to polymers containing at least two deoxyribonucleotides or ribonucleotides, either single-stranded or double-stranded, 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), self-amplified RNA, and combinations thereof. Nucleic acids include known nucleotide analogs or nucleic acids containing modified backbone residues or bindings that are synthetic, natural, and unnatural and have binding properties similar to a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methylribonucleotides, and peptide-nucleic acids (PNAs). Unless explicitly limited, this term encompasses nucleic acids containing known analogues of native nucleotides having similar binding properties to the reference nucleic acid. Unless otherwise specified, 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 substitutions can be achieved by constructing sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991), Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985), Rossolini et al., Mol. Cell. 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 their natural analogues, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that introduce new reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides.

[0150] 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.

[0151] As used herein, “gene product” refers to the product of a gene, such as an RNA transcript or polypeptide.

[0152] The term "lipids" refers to a group of organic compounds that include fatty acid esters in an unspecified manner and are insoluble in water but soluble in many organic solvents. They are usually classified into at least three classes: (1) "simple lipids" which include fats, oils, and waxes; (2) "complex lipids" which include phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0153] As used herein, the term "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 conjugate lipids to prevent particle aggregation) and nucleic acids, wherein the nucleic acid (e.g., siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, mRNA, self-amplifying RNA, or plasmids (including plasmids on which interfering RNA or mRNA is transcribed)) is encapsulated within the lipid. In one embodiment, the nucleic acid is encapsulated at least 50% within the lipid; in one embodiment, the nucleic acid is encapsulated at least 75% within the lipid; in one embodiment, the nucleic acid is encapsulated at least 90% within the lipid; and in one embodiment, the nucleic acid is encapsulated completely within the lipid. 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 extend circulatory life after intravenous (iv) injection, accumulate in distal sites (e.g., sites physically separated from the injection site), and mediate the silencing of transfected gene expression or target gene expression in these distal sites.

[0154] The lipid particles (e.g., LNPs) of the present invention typically have an average diameter of about 40 nm to about 150 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 Application Publications 20040142025 and 20070042031, which are incorporated herein by reference in their entirety for all purposes.

[0155] As used herein, “lipid-encapsulated” may refer to lipid particles that provide an activator or therapeutic agent, such as a nucleic acid (e.g., interfering RNA or mRNA), which is fully encapsulated, partially encapsulated, or both. In preferred embodiments, the nucleic acid is fully encapsulated in the lipid particle (e.g., to form SPLP, pSPLP, LNP, or other nucleic acid-lipid particles).

[0156] The term “lipid conjugate” refers to conjugated lipids that inhibit the aggregation of lipid particles. Such lipid conjugates include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), PEG-lipid conjugates such as PEG coupled to dialkyloxypropyl, PEG coupled to diacylglycerol, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, PEG conjugated to ceramide (see, for example, U.S. Patent No. 5,885,613, which is incorporated herein by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG may be conjugated directly to lipids or linked to lipids via a linker moiety. For example, any linker moiety suitable for coupling PEG to lipids can be used, including ester-free and ester-containing linker moieties. In preferred embodiments, an ester-free linker moiety is used.

[0157] The term "amphiphilic lipid" refers, in part, to any suitable substance in which the hydrophobic portion of the lipid material is oriented towards the hydrophobic phase, while the hydrophilic portion is oriented towards the aqueous phase. The hydrophilic characteristic derives from the presence of polar or charged groups such as carbohydrates, phosphoric acid, carboxyl, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other similar groups. Hydrophobicity can be conferred by including, but not limited to, nonpolar groups substituted with long-chain saturated and unsaturated aliphatic hydrocarbon groups, as well as one or more aromatic groups, alicyclic groups, or heterocyclic groups. Examples of amphiphilic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0158] Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other phosphorus-deficient compounds such as sphingolipids, the sphingoglycolipid family, diacylglycerols, and β-acyloxy acids are also included in the group designated as amphiphilic lipids. Furthermore, the aforementioned amphiphilic lipids can be mixed with other lipids, including triglycerides and sterols.

[0159] The term "neutral lipids" refers to any of several lipid species that exist in either an uncharged or neutral zwitterionic form at a given pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0160] The term "noncationic lipids" refers to any amphiphilic lipids and any other neutral or anionic lipids.

[0161] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups linked to neutral lipids.

[0162] The term "cationic lipid" refers to the compound of formula (I) as described herein.

[0163] The term "hydrophobic lipid" refers to compounds having nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups optionally substituted with one or more aromatic groups, alicyclic groups, or heterocyclic groups. Preferred examples include, but are not limited to, diacylglycerols, dialkylglycerols, NN-dialkylaminos, 1,2-diacyloxy-3-aminopropanes, and 1,2-dialkyl-3-aminopropanes.

[0164] 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.

[0165] As used herein, the term "aqueous solution" refers to a composition that contains water, either whole or in part.

[0166] As used herein, the term “organolipid solution” refers to a composition that contains, whole or in part, an organic solvent having lipids.

[0167] As used herein, "distal region" refers to a physically separated region, including not only adjacent capillary beds but also regions widely distributed throughout the entire body.

[0168] "Serum stability" for nucleic acid-lipid particles such as LNPs means that the particles are not significantly degraded after exposure to serum or nuclease assays that significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0169] As used herein, “systemic delivery” refers to the delivery of lipid particles that result in widespread biodistribution of an active or therapeutic agent, 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 such that the drug does not undergo rapid degradation or clearance (e.g., by first-pass organs (liver, lungs, etc.) or rapid nonspecific cell binding) 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.

[0170] As used herein, “local delivery” refers to the direct delivery of an activator or therapeutic agent, such as interfering RNA or mRNA, to a target site in the body. 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.

[0171] The term "mammal" refers to any species of mammal, such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, and domesticated animals.

[0172] 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 tumor conditions, whether malignant, benign, soft tissue, or solid, as well as all stages and grades of cancer, including pre-metastatic and post-metastatic cancers. 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 cancer cells.

[0173] The term "anion precursor group" includes groups that can form ions at physiological pH. For example, this term includes the groups -CO2H, -OP(=O)(OH)2, -OS(=O)2(OH), -OS(=O)(OH), and -B(OH)2. In one embodiment, the anion precursor is -CO2H.

[0174] In a particular embodiment, PEG-C-DMA has the following structure: [ka] (wherein n is selected such that the resulting polymer chain has a molecular weight of about 1000 to about 3000). In another embodiment, n is selected such that the resulting polymer chain has a molecular weight of about 2000. PEG-C-DMA can be prepared as described in Heyes et al, "Synthesis and Characterization of Novel Poly(Ethylene Glycol)-lipid Conjugates Suitable for use in Drug Delivery," Journal of Controlled Release, 2006, and U.S. Patent No. 8,936,942.

[0175] Description of the Embodiment The present invention provides novel serum-stable lipid particles comprising one or more activators or therapeutic agents, methods for producing lipid particles, and methods for delivering and / or administering lipid particles (for example, for the treatment of a disease or disorder).

[0176] In one embodiment, the present invention provides lipid particles comprising (a) one or more activators or therapeutic agents, (b) one or more cationic lipids constituting about 30 mol% to about 85 mol% of the total lipids present in the particles, (c) one or more non-cationic lipids constituting about 13 mol% to about 49.5 mol% of the total lipids present in the particles, and (d) one or more conjugate lipids that inhibit particle aggregation, constituting about 0.1 mol% to about 10 mol% of the total lipids present in the particles.

[0177] In one embodiment, the present invention provides lipid particles comprising (a) one or more activators or therapeutic agents, (b) one or more cationic lipids constituting about 50 mol% to about 85 mol% of the total lipids present in the particles, (c) one or more non-cationic lipids constituting about 13 mol% to about 49.5 mol% of the total lipids present in the particles, and (d) one or more conjugate lipids that inhibit particle aggregation, constituting about 0.5 mol% to about 2 mol% of the total lipids present in the particles.

[0178] In certain embodiments, the activator or therapeutic agent is completely encapsulated within the lipid portion of the lipid particle so that the activator or therapeutic agent in the lipid particle is resistant to enzymatic degradation, for example, by nucleases or proteases, in aqueous solution. In certain other embodiments, the lipid particle is substantially non-toxic to mammals such as humans.

[0179] In some embodiments, the activator or therapeutic agent comprises a nucleic acid. In certain cases, the nucleic acid comprises interfering RNA molecules such as siRNA, aiRNA, miRNA, or mixtures thereof. In certain other cases, the nucleic acid comprises single-stranded or double-stranded DNA, RNA, or DNA / RNA hybrids such as antisense oligonucleotides, ribozymes, plasmids, immunostimulatory oligonucleotides, or mixtures thereof. In certain cases, the nucleic acid comprises an mRNA molecule.

[0180] In other embodiments, the activator or therapeutic agent comprises a peptide or polypeptide. In certain cases, the peptide or polypeptide comprises antibodies such as, for example, polyclonal antibodies, monoclonal antibodies, antibody fragments; humanized antibodies, recombinant antibodies, recombinant human antibodies, Primatized® antibodies, or mixtures thereof. In certain other cases, the peptide or polypeptide comprises cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell surface receptors, ligands, hormones, small molecules (e.g., small organic molecules or compounds), or mixtures thereof.

[0181] In one embodiment, the activator or therapeutic agent 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 can silence the expression of a target sequence in vitro and / or in vivo.

[0182] 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%, about 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.

[0183] 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.

[0184] siRNA may contain modified nucleotides on one strand (e.g., sense or antisense) or both strands of the double-stranded region of the siRNA molecule. Preferably, uridine and / or guanosine nucleotides are modified at selective locations in the double-stranded region of the siRNA double helix. With respect to uridine nucleotide modification, at least one, two, three, four, five, six, or more of the uridine nucleotides in the sense strand and / or antisense strand may be modified uridine nucleotides, such as 2'OMe-uridine nucleotides. In some embodiments, all uridine nucleotides in the sense strand and / or antisense strand are 2'OMe-uridine nucleotides. With respect to guanosine modification, at least one, two, three, four, five, six, or more of the guanosine nucleotides in the sense strand and / or antisense strand may be modified guanosine nucleotides, such as 2'OMe-guanosine nucleotides. In some embodiments, all guanosine nucleotides in the sense strand and / or antisense strand are 2'OMe-guanosine nucleotides.

[0185] In certain embodiments, at least one, two, three, four, five, six, seven, or more 5'-GU-3' motifs of 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 on the sense strand, 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.

[0186] 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 target sequences. In other embodiments, the immunostimulant properties and ability to silence target gene expression of a modified siRNA molecule can be balanced or optimized by introducing minimal and selective 2'OMe modifications within the siRNA sequence, for example, within the double-stranded region of an siRNA double helix. In specific cases, 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 their 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).

[0187] In certain embodiments, a modified siRNA molecule is used to convert the corresponding unmodified siRNA into an IC5. 50 IC10 is less than 10 times the maximum half-dose inhibitory concentration (i.e., the maximum half-dose inhibitory concentration). 50 (i.e., modified siRNA has the IC of the corresponding unmodified siRNA) 50 ICs less than 10 times the size 50 (having). In other embodiments, the modified siRNA is IC of the corresponding unmodified siRNA sequence. 50 ICs less than 3 times the size 50 In another embodiment, the modified siRNA is the IC of the corresponding unmodified siRNA. 50 ICs less than twice the size of 50 It has a dose-response curve that can be generated using a method well known to those skilled in the art, and the IC of 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 measured.

[0188] In yet another embodiment, the modified siRNA molecule can 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.

[0189] 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.

[0190] 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.

[0191] 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 certain other 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 of the 3' end) are not modified nucleotides.

[0192] The siRNA molecules described herein may have 3' overhangs of one, two, three, four or more nucleotides on one or both sides of the double-stranded region, or 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 both sides 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 the target sequence or its complementary strand. In some embodiments, the 3' overhangs include one, two, three, four or more nucleotides, such as 2'-deoxy(2'H) nucleotides. In certain preferred embodiments, the 3' overhangs include 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 such as 2'OMe-guanosine nucleotide, 2'OMe-uridine nucleotide, 2'OMe-adenosine nucleotide, 2'OMe-cytosine nucleotide (e.g., 2'OMe purine and / or pyrimidine nucleotides), and mixtures thereof.

[0193] An 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, the cocktail contains 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 a target gene. In certain other cases, the cocktail contains 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 a target gene.

[0194] 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.

[0195] 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.

[0196] In the lipid nanoparticles of the present invention, the cationic lipid can be selected from the compounds of formula (I) described herein.

[0197] In some embodiments, cationic lipids may constitute about 30 mol% to about 90 mol%, about 30 mol% to about 85 mol%, about 30 mol% to about 80 mol%, about 30 mol% to about 75 mol%, about 30 mol% to about 70 mol%, about 30 mol% to about 65 mol%, or about 30 mol% to about 60 mol% of the total lipids present in the particles.

[0198] In some embodiments, cationic lipids may constitute about 40 mol% to about 90 mol%, about 40 mol% to about 85 mol%, about 40 mol% to about 80 mol%, about 40 mol% to about 75 mol%, about 40 mol% to about 70 mol%, about 40 mol% to about 65 mol%, or about 40 mol% to about 60 mol% of the total lipids present in the particles.

[0199] In other embodiments, cationic lipids may constitute about 55 mol% to about 90 mol%, about 55 mol% to about 85 mol%, about 55 mol% to about 80 mol%, about 55 mol% to about 75 mol%, about 55 mol% to about 70 mol%, or about 55 mol% to about 65 mol% of the total lipids present in the particles.

[0200] In yet another embodiment, cationic lipids may constitute about 60 mol% to about 90 mol%, about 60 mol% to about 85 mol%, about 60 mol% to about 80 mol%, about 60 mol% to about 75 mol%, or about 60 mol% to about 70 mol% of the total lipids present in the particles.

[0201] In further embodiments, cationic lipids may constitute approximately 65 mol% to 90 mol%, 65 mol% to 85 mol%, 65 mol% to 80 mol%, or 65 mol% to 75 mol% of the total lipids present in the particles.

[0202] In further embodiments, cationic lipids may constitute about 70 mol% to about 90 mol%, about 70 mol% to about 85 mol%, about 70 mol% to about 80 mol%, about 75 mol% to about 90 mol%, about 75 mol% to about 85 mol%, or about 80 mol% to about 90 mol% of the total lipids present in the particles.

[0203] In additional embodiments, cationic lipids may constitute (at least) about 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 mol% (or any fraction thereof or range within that).

[0204] In the lipid particles of the present invention, the noncationic lipid may include, for example, one or more anionic lipids and / or neutral lipids. In preferred embodiments, the noncationic lipid includes one of the following neutral lipid components: (1) cholesterol or a derivative thereof, (2) phospholipids, or (3) a mixture of phospholipids and cholesterol or a derivative thereof.

[0205] 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.

[0206] Phospholipids can be neutral lipids including, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylglycerol (POPG), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dierydoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), and mixtures thereof. In certain preferred embodiments, the phospholipid is DPPC, DSPC, or a mixture thereof.

[0207] In some embodiments, noncationic lipids (e.g., one or more phospholipids and / or cholesterol) may constitute about 10 mol% to about 60 mol%, about 15 mol% to about 60 mol%, about 20 mol% to about 60 mol%, about 25 mol% to about 60 mol%, about 30 mol% to about 60 mol%, about 10 mol% to about 55 mol%, about 15 mol% to about 55 mol%, about 20 mol% to about 55 mol%, about 25 mol% to about 55 mol%, about 30 mol% to about 55 mol%, about 13 mol% to about 50 mol%, about 15 mol% to about 50 mol%, or about 20 mol% to about 50 mol% of the total lipids present in the particles. If the noncationic lipids are a mixture of phospholipids and cholesterol or cholesterol derivatives, the mixture may constitute up to about 40, 50, or 60 mol% of the total lipids present in the particles.

[0208] In other embodiments, noncationic lipids (e.g., one or more phospholipids and / or cholesterol) may constitute about 10 mol% to about 49.5 mol%, about 13 mol% to about 49.5 mol%, about 15 mol% to about 49.5 mol%, about 20 mol% to about 49.5 mol%, about 25 mol% to about 49.5 mol%, about 30 mol% to about 49.5 mol%, about 35 mol% to about 49.5 mol%, or about 40 mol% to about 49.5 mol% of the total lipids present in the particles.

[0209] In yet another embodiment, noncationic lipids (e.g., one or more phospholipids and / or cholesterol) may constitute about 10 mol% to about 45 mol%, about 13 mol% to about 45 mol%, about 15 mol% to about 45 mol%, about 20 mol% to about 45 mol%, about 25 mol% to about 45 mol%, about 30 mol% to about 45 mol%, or about 35 mol% to about 45 mol% of the total lipids present in the particles.

[0210] In further embodiments, noncationic lipids (e.g., one or more phospholipids and / or cholesterol) may constitute about 10 mol% to about 40 mol%, about 13 mol% to about 40 mol%, about 15 mol% to about 40 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 40 mol%, or about 30 mol% to about 40 mol% of the total lipids present in the particles.

[0211] In further embodiments, noncationic lipids (e.g., one or more phospholipids and / or cholesterol) may constitute about 10 mol% to about 35 mol%, about 13 mol% to about 35 mol%, about 15 mol% to about 35 mol%, about 20 mol% to about 35 mol%, or about 25 mol% to about 35 mol% of the total lipids present in the particles.

[0212] In further embodiments, noncationic lipids (e.g., one or more phospholipids and / or cholesterol) may constitute about 10 mol% to about 30 mol%, about 13 mol% to about 30 mol%, about 15 mol% to about 30 mol%, about 20 mol% to about 30 mol%, about 10 mol% to about 25 mol%, about 13 mol% to about 25 mol%, or about 15 mol% to about 25 mol% of the total lipids present in the particles.

[0213] In additional embodiments, noncationic lipids (e.g., one or more phospholipids and / or cholesterol) may constitute (at least) about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol% (or any fraction thereof or range within that).

[0214] In certain preferred embodiments, the noncationic lipids comprise about 31.5 mol% to about 42.5 mol% of cholesterol or its derivatives of the total lipids present in the particles. As a non-limiting example, the phospholipid-free lipid particles of the present invention may comprise about 37 mol% of cholesterol or its derivatives of the total lipids present in the particles. In other preferred embodiments, the phospholipid-free lipid particles of the present invention may contain about 30 mol% to about 45 mol%, about 30 mol% to about 40 mol%, about 30 mol% to about 35 mol%, about 35 mol% to about 45 mol%, about 40 mol% to about 45 mol%, about 32 mol% to about 45 mol%, about 32 mol% to about 42 mol%, about 32 mol% to about 40 mol%, about 34 mol% to about 45 mol%, about 34 mol% to about 42 mol%, about 34 mol% to about 40 mol%, or about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 mol% (or any fraction thereof or a range within thereof) of cholesterol or a derivative thereof of the total lipids present in the particles.

[0215] In certain other preferred embodiments, the noncationic lipids include a mixture of (i) phospholipids in an amount of about 4 mol% to about 10 mol% of the total lipids present in the particles, and (ii) cholesterol or its derivatives in an amount of about 30 mol% to about 40 mol% of the total lipids present in the particles. As a non-limiting example, lipid particles containing a mixture of phospholipids and cholesterol may include about 7 mol% DPPC and about 34 mol% cholesterol in an amount of the total lipids present in the particles. In other embodiments, the noncationic lipids are (i) about 3 mol% to about 15 mol%, about 4 mol% to about 15 mol%, about 4 mol% to about 12 mol%, about 4 mol% to about 10 mol%, about 4 mol% to about 8 mol%, about 5 mol% to about 12 mol%, about 5 mol% to about 9 mol%, about 6 mol% to about 12 mol%, about 6 mol% to about 10 mol%, or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% (or any fraction thereof or range within that) of the total lipids present in the particles, and (ii) about 25 mol% to about 45 mol%, about 30 mol% to about 45 mol% of the total lipids present in the particles. It contains a mixture of 1%, approximately 25 mol% to approximately 40 mol%, approximately 30 mol% to approximately 40 mol%, approximately 25 mol% to approximately 35 mol%, approximately 30 mol% to approximately 35 mol%, approximately 35 mol% to approximately 45 mol%, approximately 40 mol% to approximately 45 mol%, approximately 28 mol% to approximately 40 mol%, approximately 28 mol% to approximately 38 mol%, approximately 30 mol% to approximately 38 mol%, approximately 32 mol% to approximately 36 mol%, or approximately 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 mol% (or any fraction thereof or range within that) of cholesterol or its derivatives.

[0216] In a more preferred embodiment, the noncationic lipids include a mixture of (i) phospholipids in an amount of about 10 mol% to about 30 mol% of the total lipids present in the particles, and (ii) cholesterol or a derivative thereof in an amount of about 10 mol% to about 30 mol% of the total lipids present in the particles. As a non-limiting example, lipid particles containing a mixture of phospholipids and cholesterol may contain about 20 mol% DPPC and about 20 mol% cholesterol in an amount of the total lipids present in the particles. In other embodiments, the noncationic lipids are (i) about 10 mol% to about 30 mol%, about 10 mol% to about 25 mol%, about 10 mol% to about 20 mol%, about 15 mol% to about 30 mol%, about 20 mol% to about 30 mol%, about 15 mol% to about 25 mol%, about 12 mol% to about 28 mol%, about 14 mol% to about 26 mol%, or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mol% (or any fraction thereof or a range within thereof) of the total lipids present in the particles, (ii) A mixture of cholesterol or its derivatives comprising approximately 10 mol% to approximately 30 mol%, approximately 10 mol% to approximately 25 mol%, approximately 10 mol% to approximately 20 mol%, approximately 15 mol% to approximately 30 mol%, approximately 20 mol% to approximately 30 mol%, approximately 15 mol% to approximately 25 mol%, approximately 12 mol% to approximately 28 mol%, approximately 14 mol% to approximately 26 mol%, or approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mol% (or any fraction thereof or a range within thereof) of the total lipids present in the particles.

[0217] Conjugate lipids In the lipid particles of the present invention (for example, LNPs (including interfering RNA such as siRNA, or mRNA)), the conjugate lipid may include, for example, one or more of the following: polyethylene glycol (PEG)-lipid conjugate, polyamide (ATTA)-lipid conjugate, or mixtures thereof. In a preferred embodiment, the nucleic acid-lipid particle includes either a PEG-lipid conjugate or an ATTA-lipid conjugate. The conjugate lipid may include, for example, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmityloxypropyl (C16), PEG-distearyloxypropyl (C18), or mixtures thereof.

[0218] Further PEG-lipid conjugates suitable for use in the present invention include, but are not limited to, mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG). The synthesis of PEG-C-DOMG is described in PCT application PCT / US08 / 88676, filed on December 31, 2008, and this disclosure is incorporated herein by reference in its entirety for all purposes. Further PEG-lipid conjugates suitable for use in the present invention include, but are not limited to, 1-[8'-(1,2-dimiristoyl-3-propaneoxy)-carboxamide-3',6'-dioxaoctanyl]carbamoyl-w-methyl-poly(ethylene glycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S. Patent No. 7,404,969, which is incorporated herein by reference in its entirety for all purposes.

[0219] The PEG portion of the PEG-lipid conjugates described herein may have an average molecular weight in the range of about 550 daltons to about 10,000 daltons. In specific cases, the PEG portion has an average molecular weight of about 750 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG portion has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0220] In certain cases, conjugated lipids (e.g., PEG-lipid conjugates) may constitute approximately 0.1 to 10% (or any fraction thereof or within that range) of the total lipids present in the particles. In certain cases, conjugated lipids (e.g., PEG-lipid conjugates) may constitute approximately 0.1 mol% to 2 mol%, 0.5 mol% to 2 mol%, 1 mol% to 2 mol%, 0.6 mol% to 1.9 mol%, 0.7 mol% to 1.8 mol%, 0.8 mol% to 1.7 mol%, 1 mol% to 1.8 mol%, 1.2 mol% to 1.8 mol%, 1.2 mol% to 1.7 mol%, 1.3 mol% to 1.6 mol%, 1.4 mol% to 1.5 mol%, or approximately 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol% (or any fraction thereof or a range within that range) of the total lipids present in the particles.

[0221] In the lipid particles of the present invention, the activator or therapeutic agent is completely encapsulated within the lipid portion of the particle, thereby protecting the activator or therapeutic agent from nuclease 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 acid from nuclease degradation. In certain cases, the nucleic acid in the LNP remains substantially undegraded after the particle is exposed to a nuclease at 37°C for at least about 20, 30, 45, or 60 minutes. In certain other cases, the nucleic acid in the LNP remains substantially undegraded after the particle is 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, an activator or therapeutic agent (e.g., a nucleic acid such as siRNA) forms a complex with the lipid portion of the particle. One advantage of the formulations of the present invention is that the lipid particle composition is substantially non-toxic to mammals such as humans.

[0222] The term "fully encapsulated" indicates that the activator or therapeutic agent in the lipid particles is 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 the free activator or therapeutic agent, preferably less than about 25% of the activator or therapeutic agent in the particles is degraded, more preferably less than about 10%, and most preferably less than about 5%. In relation to nucleic acid therapeutic agents, fully encapsulated can be determined by the Oligreen® assay. Oligreen® is an ultra-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 serum-stable, i.e., they are not rapidly degraded into their components upon in vivo administration.

[0223] In another aspect, the present invention provides a lipid particle (e.g., LNP) composition comprising a plurality of lipid particles. In a preferred embodiment, the activator or therapeutic agent (e.g., nucleic acid) is present in concentrations of 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%. %, 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).

[0224] 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. In preferred embodiments, the lipid particles of the present invention have a lipid:activator (e.g., lipid:nucleic acid) ratio (mass / mass ratio) of about 5 to about 15, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (or any fraction thereof or range within that range).

[0225] Typically, the lipid particles (e.g., LNPs) of the present invention have an average diameter of about 40 nm to about 150 nm. In a preferred embodiment, the lipid particles (e.g., LNPs) 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 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 less than about 120 nm, less than about 110 nm, less than about 100 nm, less than about 90 nm, or less than about 80 nm (or any fraction thereof or a range within that).

[0226] In a particular embodiment of the present invention, the LNP comprises (a) one or more unmodified and / or modified nucleic acid molecules (e.g., interfering RNAs that silence the expression of target genes, such as siRNA, aiRNA, miRNA, or mRNA, or mRNAs that cause the expression of target proteins), (b) cationic lipids constituting about 56.5 mol% to about 66.5 mol% of the total lipids present in the particles, (c) non-cationic lipids constituting about 31.5 mol% to about 42.5 mol% of the total lipids present in the particles, and (d) conjugate lipids that inhibit particle aggregation constituting about 1 mol% to about 2 mol% of the total lipids present in the particles. This particular embodiment of the LNP is generally referred to herein as the “1:62” formulation. In preferred embodiments, the cationic lipid is DLinDMA or DLin-K-C2-DMA (“XTC2”), the non-cationic lipid is cholesterol, and the conjugate lipid is a PEG-DAA conjugate. These are preferred embodiments of the 1:62 formulation, but those skilled in the art will understand that other cationic lipids, non-cationic lipids (including other cholesterol derivatives), and conjugate lipids can be used in the 1:62 formulation described herein.

[0227] In another specific embodiment of the present invention, the LNP comprises (a) one or more unmodified and / or modified nucleic acid molecules (e.g., interfering RNAs that silence the expression of target genes, such as siRNA, aiRNA, miRNA, or mRNA, or mRNAs that cause the expression of target proteins), (b) cationic lipids constituting about 52 mol% to about 62 mol% of the total lipids present in the particles, (c) non-cationic lipids constituting about 36 mol% to about 47 mol% of the total lipids present in the particles, and (d) conjugate lipids that inhibit particle aggregation constituting about 1 mol% to about 2 mol% of the total lipids present in the particles. This particular embodiment of the LNP is generally referred to herein as the “1:57” formulation. In one preferred embodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA ("XTC2"), the non-cationic lipid is a mixture of phospholipids (such as DPPC) and cholesterol (where the phospholipids constitute about 5 mol% to about 9 mol% (e.g., about 7.1 mol%) of the total lipids present in the particles, and the cholesterol (or cholesterol derivatives) constitute about 32 mol% to about 37 mol% (e.g., about 34.3 mol%) of the total lipids present in the particles), and the PEG-lipid is PEG-DAA (e.g., PEG-cDMA). In another preferred embodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA ("XTC2"), the non-cationic lipid is a mixture of phospholipid (such as DPPC) and cholesterol (where the phospholipid constitutes about 15 mol% to about 25 mol% (e.g., about 20 mol%) of the total lipids present in the particles, and the cholesterol (or cholesterol derivative) constitutes about 15 mol% to about 25 mol% (e.g., about 20 mol%) of the total lipids present in the particles), and the PEG-lipid is PEG-DAA (e.g., PEG-cDMA). These are preferred embodiments of the 1:57 formulation, but those skilled in the art will understand that other cationic lipids, non-cationic lipids (including other phospholipids and other cholesterol derivatives), and conjugate lipids can be used in the 1:57 formulation described herein.

[0228] In a preferred embodiment, the 1:62 LNP formulation is a three-component system that does not contain phospholipids and consists of about 1.5 mol% PEG-cDMA (or PEG-IDSA), about 61.5 mol% DLinDMA (or XTC2), and about 36.9 mol% cholesterol (or its derivative). In another preferred embodiment, the 1:57 LNP formulation is a four-component system consisting of about 1.4 mol% PEG-cDMA (or PEG-cDSA), about 57.1 mol% DLinDMA (or XTC2), about 7.1 mol% DPPC, and about 34.3 mol% cholesterol (or its derivative). In yet another preferred embodiment, the 1:57 LNP formulation is a four-component system consisting of about 1.4 mol% PEG-cDMA (or PEG-cDSA), about 57.1 mol% DLinDMA (or XTC2), about 20 mol% DPPC, and about 20 mol% cholesterol (or its derivative). These LNP formulations are target formulations, and it should be understood that the amount of lipids (both cationic and non-cationic) and lipid conjugates present in the LNP formulations may vary.

[0229] The present invention also provides pharmaceutical compositions comprising lipid particles (e.g., LNPs) as described herein and a pharmaceutically acceptable carrier.

[0230] In further embodiments, the present invention provides a method for introducing one or more activators 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 present 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 activators 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.

[0231] 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 even up to 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 multiple 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 an activator or therapeutic agent, such as interfering RNA (e.g., siRNA) or mRNA, is detectable intracellularly about 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 an activator or therapeutic agent, such as interfering RNA (e.g., siRNA), is detectable about 8, 12, 24, 36, 48, 60, 72, or 96 hours after administration. In yet another embodiment, downregulation of target sequence expression by an activator or therapeutic agent, 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 an activator or therapeutic agent, 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 an activator or therapeutic agent, 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 an activator or therapeutic agent, 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 activators or therapeutic agents, such as mRNA or self-replicating RNA, 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 additional embodiments, the lipid particles of the present invention (e.g., LNPs) are administered parenterally or intraperitoneally.

[0232] In some embodiments, the lipid particles (e.g., LNPs) of the present invention are particularly 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 certain other embodiments, a therapeutically effective amount of lipid particles (e.g., LNPs) can be administered to a mammal. In some cases, interfering RNA (e.g., siRNA) is formulated into 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.

[0233] 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.

[0234] In one embodiment, the aiRNA molecule comprises a double-stranded region of approximately 10 to 25 (base-pairing) nucleotides in length, and the aiRNA molecule includes an antisense strand with 5' and 3' overhangs, and the aiRNA molecule can silence target gene expression.

[0235] In one embodiment, the aiRNA molecule includes a double-stranded region of approximately 12–20, 12–19, 12–18, 13–17, or 14–17 (base-pairing) nucleotide lengths, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 (base-pairing) nucleotide lengths. In certain other cases, the 5' and 3' overhangs on the antisense strand include sequences complementary to the target RNA sequence and may optionally further include non-target sequences. 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.

[0236] 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.

[0237] 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.

[0238] In one embodiment, the miRNA molecule consists of approximately 15 to 60 nucleotides in length, and the miRNA molecule can silence the expression of a target gene.

[0239] In certain cases, miRNA molecules consist of approximately 15–50, 15–40, or 15–30 nucleotides in length, 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.

[0240] 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.

[0241] In some embodiments, the lipid particles (e.g., LNPs) of the present invention are useful for methods of 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 treating 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. In non-limiting examples, the methods of the present invention are useful for in vivo delivery of one or more mRNA molecules to mammalian subjects. In certain other embodiments, a therapeutically effective amount of lipid particles (e.g., LNPs) can be administered to a mammal. In some cases, one or more mRNA molecules are formulated into LNPs and these particles are administered to a patient in need of such treatment. In other cases, cells are taken from a 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.

[0242] In other embodiments, the mRNA 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.

[0243] Therefore, the lipid particles (e.g., LNPs) of the present invention are 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) to subjects (e.g., mammals such as humans) because they are stable in circulation, are of a size required for pharmacodynamic behavior that provides access to extravascular sites, and can reach target cell populations.

[0244] Activating agent Activators (e.g., therapeutic agents) include any molecule or compound that can exert a desired effect on cells, tissues, organs, or targets. Such effects may be, for example, biological, physiological, and / or cosmetic effects. Activators can be any type of molecule or compound, including but not limited to nucleic acids, peptides, polypeptides, small molecules, and mixtures thereof. Non-limited examples of nucleic acids include interfering RNA molecules (e.g., siRNA, aiRNA, miRNA), antisense oligonucleotides, mRNA, auto-amplified RNA, plasmids, ribozymes, immunostimulatory oligonucleotides, and mixtures thereof. Examples of peptides or polypeptides include, but are not limited to, antibodies (e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments; humanized antibodies, recombinant antibodies, recombinant human antibodies, Primatized® antibodies), cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell surface receptors and their ligands, hormones, and mixtures thereof. Examples of small molecules include, but are not limited to, organic small molecules or compounds such as any conventional drugs or medications known to those skilled in the art.

[0245] In some embodiments, the activator is a therapeutic agent, a salt thereof, or a derivative thereof. The therapeutic agent derivative may be therapeutically active in itself, or it may be a prodrug that becomes active when further modified. Thus, in one embodiment, the therapeutic agent derivative retains some or all of the therapeutic activity compared to the unmodified agent, while in another embodiment, the therapeutic agent derivative is a prodrug that lacks therapeutic activity but becomes active when further modified.

[0246] nucleic acid In certain embodiments, the lipid particles of the present invention associate with nucleic acids to form nucleic acid-lipid particles (e.g., LNPs). In some embodiments, the nucleic acids are completely encapsulated within the lipid particles. As used herein, the term “nucleic acid” includes any oligonucleotide or polynucleotide having fragments containing up to 60 nucleotides, commonly referred to as oligonucleotides, and longer fragments referred to as polynucleotides. In certain embodiments, the oligonucleotides of the present invention are about 15 to about 60 nucleotides long. Nucleic acids can be administered alone with the lipid particles of the present invention, or in combination with (e.g., in combination with) the lipid particles of the present invention containing low molecules such as peptides, polypeptides, or conventional drugs.

[0247] In the context of this invention, the terms “polynucleotide” and “oligonucleotide” refer to polymers or oligomers of nucleotides or nucleoside monomers consisting of natural bases, sugars, and intersugar (skeletal) bonds. The terms “polynucleotide” and “oligonucleotide” also include polymers or oligomers containing similarly functioning non-natural monomers or parts thereof. Such modified or substituted oligonucleotides are often preferred over the natural form due to properties such as improved cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0248] Oligonucleotides are generally classified into deoxyribooliknucleotides or ribooliknucleotides. Deoxyribooliknucleotides consist of a five-carbon sugar called deoxyribose, which is covalently bonded to phosphate at its 5' and 3' carbon atoms, forming alternating unbranched polymers. Ribooliknucleotides consist of a similar repeating structure in which the five-carbon sugar is ribose.

[0249] 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.

[0250] The nucleic acids of the present invention may 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 nucleotide residues long. In certain embodiments, an oligonucleotide may be in the range of about 10 to about 100 nucleotides long. In various related embodiments, single-stranded, double-stranded, and triple-stranded oligonucleotides may be in the range of about 10 to about 60 nucleotides long, about 15 to about 60 nucleotides long, about 20 to about 50 nucleotides long, about 15 to about 30 nucleotides long, or about 20 to about 30 nucleotides long.

[0251] 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 degree of complementarity sufficient to result in stable and specific binding between the DNA or RNA target 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, if binding of the oligonucleotide to the target sequence interferes with the normal function of the target sequence, resulting in a loss of usefulness or expression from the target sequence, then the oligonucleotide is specifically hybridizable and has a degree of complementarity sufficient to avoid nonspecific binding of the oligonucleotide to non-target sequences under conditions where specific binding is desired, for example, 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, the 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.

[0252] siRNA The siRNA component of the nucleic acid-lipid particles of the present invention can silence the expression of a target gene of interest. Each strand of the siRNA double helix is ​​typically about 15 to about 60 nucleotides long, preferably about 15 to about 30 nucleotides long. In certain embodiments, the siRNA contains at least one modified nucleotide. Modified siRNAs are generally less immunostimulant than the corresponding unmodified siRNA sequence 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, e.g., 2'OMe-guanosine nucleotide, 2'OMe-uridine nucleotide, 2'OMe-adenosine nucleotide, and / or 2'OMe-cytosine nucleotide. In preferred embodiments, one or more of the uridine and / or guanosine nucleotides are modified. The modified nucleotide may be present on one strand of the siRNA (e.g., sense or antisense) or on both strands. 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 not have an overhang (i.e., it may have a blunt end).

[0253] Modified siRNA generally contains approximately 1% to 100% (e.g., approximately 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 double helix. In certain embodiments, one, two, three, four, five, six, seven, eight, nine, ten, or more nucleotides within the double-stranded region of the siRNA include modified nucleotides.

[0254] In some embodiments, less than 25% of the nucleotides in the double-stranded region of the siRNA (e.g., less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%) consist of modified nucleotides.

[0255] In other embodiments, approximately 1% to 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 20% (e.g., approximately 1% to 2%). 0%, 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%~18% %, 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%~ Percentages such as 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% include modified nucleotides.

[0256] In a further embodiment, for example, if one or both strands of the siRNA are selectively modified in uridine and / or guanosine nucleotides, the resulting modified siRNA has less than 30% modified nucleotides (e.g., less than 30%, less than 29%, less than 28%, less than 27%, less than 26%, less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2 (less than 1%, or approximately less than 1% modified nucleotides), or approximately 1% to approximately 30% modified nucleotides (for example, approximately 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%, 1 It may contain modified nucleotides of 4%-30%, 15%-30%, 16%-30%, 17%-30%, 18%-30%, 19%-30%, 20%-30%, 21%-30%, 22%-30%, 23%-30%, 24%-30%, 25%-30%, 26%-30%, 27%-30%, 28%-30%, or 29%-30%.

[0257] 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).

[0258] 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)). The nucleotides immediately 3' of the dinucleotide sequence are identified as potential siRNA sequences (e.g., target sequence or sense strand sequence). Typically, 19, 21, 23, 25, 27, 29, 31, 33, 35, or more nucleotides immediately 3' of the dinucleotide sequence are identified as potential siRNA sequences. In some embodiments, the dinucleotide sequence is an AA or NA sequence, and the 19 nucleotides immediately 3' of the AA or NA dinucleotide are identified as potential siRNA sequences. The siRNA sequences are generally spaced apart at different positions along the length of the target gene. To further enhance the silencing efficiency of siRNA sequences, potential siRNA sequences can be analyzed to identify regions that do not contain homologous areas with other coding sequences in, for example, target cells or organisms. For instance, 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 PolIII promoter, select siRNA sequences that lack more than four consecutive A or T base pairs.

[0259] After identifying potential siRNA sequences, complementary sequences (e.g., 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 can be analyzed using rational design algorithms to identify sequences having one or more of the following characteristics: (1) a 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 having one or more of the aforementioned characteristics can be selected as potential siRNA sequences for further analysis and testing.

[0260] Furthermore, potential siRNA sequences having one or more of the following criteria are often excluded as siRNA: (1) sequences containing four or more consecutive identical bases, (2) sequences containing G homopolymers (e.g., to reduce possible nonspecific effects due to the structural properties of these polymers), (3) sequences containing triple base motifs (e.g., GGG, CCC, AAA, or TTT), (4) sequences containing seven or more consecutive G / C, and (5) sequences containing four or more direct repeats of bases within the candidate, resulting in an internal foldback structure. However, those skilled in the art will understand that sequences having one or more of the aforementioned features can still be selected as potential siRNA sequences for further analysis and testing.

[0261] 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 that are favorable for target site reachability, with minimal secondary structure in the form of base pairings and the presence of stem-loops.

[0262] After identifying potential siRNA sequences, the sequences can be analyzed for the presence of any immunostimuliable properties, 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 originate from naive mammals (i.e., mammals that have never been in contact with the gene product of the siRNA sequence). Mammalian responder cells may include, 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. Next, an siRNA molecule identified as immunostimulant can be modified by substituting at least one of the nucleotides in the sense strand and / or antisense strand with a modified nucleotide to reduce its immunostimulant properties. 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 have been reduced or suppressed.

[0263] Suitable in vitro assays 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, for example, by 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.

[0264] 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 assay can be carried out 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.)).

[0265] 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.

[0266] Production of siRNA molecules siRNA can be provided in several forms, including, for example, as one or more isolated small interfering RNA (siRNA) double helix, 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 may lack overhangs (i.e., to have blunt ends).

[0267] Long precursor RNA can be obtained using RNA populations, or siRNA can be constructed using long precursor RNA that has substantial or complete identity with respect to a selected target sequence. RNA can be isolated, synthesized, and / or cloned from cells or tissues according to methods well known to those skilled in the art. RNA can be a mixed population (obtained from cells or tissues, transcribed from cDNA, subtracted, selected, etc.) or can represent a single target sequence. RNA can be natural (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.

[0268] To form long dsRNAs, in the case of synthetic RNA, the complement is also transcribed and hybridized in vitro to form the dsRNA. When using natural RNA populations, RNA complements are also provided (for example, to form dsRNAs for digestion by E. coli RNAseIII or Dicer) by transcribing cDNA corresponding to the RNA population or by using RNA polymerase. The precursor RNA is then hybridized to form double-stranded RNA for digestion. The dsRNA can be administered directly to the target or digested in vitro before administration.

[0269] 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 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), Sambrook et al., cited above, and Ausubel et al., cited above). 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.

[0270] Preferably, the siRNA is chemically synthesized. Oligonucleotides containing the siRNA molecule of the present invention can be synthesized using any of the 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 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 in 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 96-well plate synthesizer from Protogene (Palo Alto, Calif.). However, synthesis on larger or smaller scales is also within the scope of the present invention. Reagents suitable for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those skilled in the art.

[0271] siRNA molecules can also be synthesized via tandem synthesis techniques, where both strands are synthesized as a single contiguous oligonucleotide fragment or chain separated by a cleavable linker, which is then cleaved to yield 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 assembled from two separate oligonucleotides, one containing the sense strand of the siRNA and the other containing the antisense strand. For example, each strand can be synthesized separately and then linked together by hybridization or ligation after synthesis and / or deprotection. In certain other cases, an siRNA molecule can be synthesized as a single contiguous oligonucleotide fragment, where the self-complementary sense and antisense regions hybridize to form an siRNA double helix with a hairpin secondary structure.

[0272] 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.

[0273] 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 containing nucleotide base analogs, such as C-phenyl and C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, 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 siRNA molecules.

[0274] In certain embodiments, the siRNA molecule may further include one or more chemical modifications, such as terminal capping moieties, phosphate backbone modifications, etc. Examples of terminal capping moieties 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 moieties, 3'-3'-inverted debaseping moieties, 3'-2'-inverted nucleotide moieties, 3'-2'-inverted debaseping moieties, 5'-5'-inverted nucleotide moieties, 5'-5'-inverted debaseping moieties, 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'-phosphoroamidates, 5'-phosphoroamidates, 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 (e.g., resulting in modified internucleotide bonds) include phosphorothioates, phosphorodithioates, methylphosphonates, phosphotriesters, morpholinos, amideates, carbamates, carboxymethyl acetamides, polyamides, sulfonates, sulfonamides, sulfamates, formacetals, thioformacetals, and alkylsilyl substitutions (see, e.g., Hunziker et al., Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417 (1995); Mesmaeker et al., Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39 (1994)). Such chemical modifications can occur at the 5' and / or 3' ends of the sense strand, antisense strand, or both strands of an siRNA. The disclosures of these references are incorporated herein by reference in their entirety for all purposes.

[0275] In some embodiments, the sense and / or antisense strands of the siRNA molecule may further include a 3' terminal overhang having about one to about four (e.g., one, two, three, or four) 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 an siRNA molecule are described, for example, in UK Patent GB2,397,818B and U.S. Patent Applications Publications 20040192626, 20050282188, and 20070135372, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0276] 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 that can be incorporated into a nucleic acid chain in place of one or more nucleotide units, including sugar and / or phosphate substitutions, 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.

[0277] In other embodiments, the chemical modification of siRNA involves 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, for example, via a covalent bond such as a biodegradable linker. The conjugate may also be attached to the siRNA, for example, via a carbamate group or other linking group (see, for example, U.S. Patent Applications 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 cellular uptake, and combinations thereof (see, for example, U.S. Patent Applications Publications 20030130186, 20040110296, and 20040249178, and U.S. Patent No. 6,753,423). Other examples include lipophilic moieties, vitamins, polymers, peptides, proteins, nucleic acids, low molecular weight molecules, oligosaccharides, carbohydrate clusters, intercalators, sub-groove binders, cleavage agents, and crosslinking agent conjugate molecules, as described in U.S. Patent Application 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 Application 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 Application Publication No. 20040167090. Further examples include conjugate molecules described in U.S. Patent Application Publication No. 20050239739. The type of conjugate used and the degree of conjugation with the siRNA molecule can be evaluated for the improved pharmacokinetic profile, bioavailability, and / or stability of the siRNA while maintaining RNAi activity. Thus, those skilled in the art can use any of the various well-known in vitro cell cultures or in vivo animal models to screen siRNA molecules bound to various conjugates and identify those with improved properties and full RNAi activity. The disclosures of the aforementioned patent documents are incorporated herein by reference in their entirety for all purposes.

[0278] target genes In certain embodiments, the nucleic acid component (e.g., siRNA) of the nucleic acid-lipid particles described herein 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.

[0279] Genes related to viral infection and survival include those expressed by the virus to bind to, enter, 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)).

[0280] 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 accession numbers 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 accession numbers 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 the Marburg virus is described, for example, in Genbank accession numbers NC_001608, AY430365, AY430366, and AY358025. The sequence of the 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 the nucleic acid sequences of Ebola virus and Marburg virus include those described in U.S. Patent Application Publication 20070135370, which is incorporated herein by reference in its entirety for all purposes.

[0281] 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, This is described in AY651497, AY651498, AY651499, AY651500, AY651501, AY651502, AY651503, AY651504, AY651505, AY651506, AY651507, AY651509, AY651528, AY770996, AY790308, AY818138, and AY818140. The PA sequences of influenza A are, for example, Genbank accession numbers AY818132, AY790280, AY646171, AY818132, AY818133, AY646179, AY818134, AY551934, AY651613, AY651610, AY651620, AY651617, AY651600, A These are described in Y651611, 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 Application Publication No. 20070218122, which is incorporated herein by reference in its entirety for all purposes.

[0282] 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 Applications Publications 20060281175, 20050058982, and 20070149470, U.S. Patent No. 7,348,314, and U.S. Provisional Patent Application No. 61 / 162,127 filed March 20, 2009, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0283] 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 Application 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 January 26, 2009, which is incorporated herein by reference in its entirety for all purposes.

[0284] 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_00683) 7) 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 include those described in U.S. Patent Application No. 11 / 807,872, filed on 29 May 2007, which is incorporated herein by reference in its entirety for all purposes. Non-limiting examples of siRNA molecules targeting the PLK-1 gene include those described in U.S. Patent 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 all purposes. 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 all purposes.

[0285] 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) These include mutant sequences such as Res., 285:39-49 (2003), 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 RAS (described 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, which is incorporated herein by reference in its entirety for all purposes.

[0286] 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.

[0287] 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 Application Publication No. 20040142895, and CA2456444, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0288] 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.

[0289] 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.

[0290] Cell receptor ligands include ligands that bind to cell surface receptors (e.g., insulin receptors, EPO receptors, G protein-coupled receptors, tyrosine kinase receptors, cytokine receptors, growth factor receptors, 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 (e.g., CAG repeats) elongation are used to silencing 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)).

[0291] 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-HS- Glycoprotein (AHSG), aldo-ketoreductase 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), apolipoprotein 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).

[0292] 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.

[0293] 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 clustered, regularly spaced, short palindromic repetition (CRISPR) technology (see, e.g., Sander et al., Nature Biotechnology, 32(4), 347-355, including Supplementary Information (2014), and International Publications WO2016 / 197132 and 2016 / 197133). Therefore, provided herein are improvements (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.

[0294] aiRNA Similar to siRNA, asymmetric interfering RNA (aiRNA) can effectively silence various genes in mammalian cells by recruiting the RNA-induced silencing complex (RISC) and mediating sequence-specific cleavage of a target sequence between nucleotides 10 and 11 relative to the 5' end of the antisense strand (Sun et al., Nat. Biotech., 26:1379-1382 (2008)). Typically, aiRNA molecules contain a short RNA double helix with a sense strand and an antisense strand, and this double helix contains overhangs at the 3' and 5' ends of the antisense strand. aiRNA is 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.

[0295] In another embodiment, aiRNA double helix 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 20 base pairs) can be designed to include overhangs at the 3' and 5' ends of the antisense strand to target the mRNA of interest. 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 certain other 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.

[0296] 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.

[0297] 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 can 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 disorders.

[0298] miRNA Generally, microRNAs (miRNAs) are single-stranded RNA molecules, approximately 21-23 nucleotides long, that regulate gene expression. miRNAs are encoded by genes, and are transcribed from the DNA of those genes. However, miRNAs 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, and finally 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.

[0299] The genes encoding miRNAs are much longer than the processed mature miRNA molecules. miRNAs are first transcribed as primary transcripts or pri-miRNAs, which have a cap and poly(A) tail. In the cell nucleus, these are processed into short stem-loop structures of about 70 nucleotides known as pre-miRNAs. This processing occurs in animals by a protein complex known as the microprocessor complex, consisting of a nuclease drothera 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 endonuclease dicers, 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 function as a template for generating miRNAs.

[0300] When a 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 degradation of the target mRNA and / or silencing of its translation.

[0301] Mammalian miRNA molecules are typically complementary to a site within the 3' UTR of the target mRNA sequence. In certain cases, annealing of miRNA to target mRNA inhibits protein translation by blocking the protein translation mechanism. In certain other cases, annealing of miRNA to 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.

[0302] 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.

[0303] In some embodiments, miRNA molecules can 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 disorders.

[0304] In other embodiments, one or more agents that block the activity of miRNAs targeting the mRNA of interest are administered using lipid particles of the present invention (e.g., nucleic acid-lipid particles). 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.

[0305] 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 nucleotide 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 non-target-specific 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.

[0306] 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, 5,789,573, 5,718,709, and 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.

[0307] 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 is performed by analyzing 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 can 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)).

[0308] Ribozyme According to another embodiment of the present invention, nucleic acid-lipid particles are associated 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-pair interaction before the chemical reaction.

[0309] 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 a new target.

[0310] 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.

[0311] Methods for generating ribozymes targeting arbitrary polynucleotide sequences are known in the art. Ribozymes can 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.

[0312] 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, which 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 all purposes), modifications that enhance their efficacy in cells, and removal of stem II bases for shortening RNA synthesis time and reducing chemical requirements.

[0313] 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, which is incorporated herein by reference in its entirety for all purposes).

[0314] Immunostimulatory nucleic acids are considered sequence-specific 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 regions of native genes or mRNA, but they can still be considered sequence-specific immunostimulatory nucleic acids.

[0315] 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), each of which is incorporated herein by reference in its entirety for all purposes. 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 within a CpG motif.

[0316] mRNA In certain embodiments, the nucleic acid is one or more mRNA molecules (e.g., a cocktail of mRNA molecules).

[0317] 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 cell into which the mRNA is introduced compared with the corresponding unmodified mRNA.

[0318] 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 thio-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseuduridine, 4-thio-1-methyl-pseuduridine, 2-thio-1-methyl-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.

[0319] 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.

[0320] 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.

[0321] 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 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.

[0322] 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.

[0323] 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.

[0324] Optional 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.

[0325] Furthermore, nucleic acids containing one or more intronic nucleotide sequences that can be cleaved from the nucleic acid are provided.

[0326] 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 that the downregulation of the transcript is controlled in the event of misdirection to an undesirable organ site.

[0327] 5' capping The 5' cap structure of mRNA is involved in nuclear export, increases mRNA stability, and binds to mRNA cap-binding proteins (CBPs). This CBPs, through association with poly(A)-binding proteins, contribute to the stability and translational capacity of mRNA within the cell, forming mature circular mRNA species. The cap also assists in the removal of the 5' proximal intron during mRNA splicing.

[0328] The endogenous mRNA molecule may be capped at its 5' end, creating a 5'-ppp-5'-triphosphate bond between the terminal guanosine cap residue and the transcribed sense nucleotide at the 5' end of the mRNA molecule. 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 nucleotide of the mRNA may also be optionally 2'-O-methylated. Removal of the 5'-cap by hydrolysis and cleavage of the guanylate cap structure may target nucleic acid molecules such as mRNA molecules for degradation.

[0329] 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 multiple 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 (S1V), or cricket paralysis virus (CrPV).

[0330] 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.

[0331] 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).

[0332] 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 the portion 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.

[0333] 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.

[0334] Coded polypeptide The mRNA component of the nucleic acid-lipid particles described herein can be used to express the target polypeptide. 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 encoding gene, or due to mutations in the encoding 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 genes encoding common gamma-chain proteins, which are components 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.

[0335] 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.

[0336] 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 immune responses to therapies. 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 LNPs, compositions and methods of the present invention may be useful, for example, in the delivery or expression of vaccine antigens for the treatment of cancer.

[0337] 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 can 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 the RNA-dependent RNA polymerase (replicase) protein and the second ORF encodes a structural protein. In a sa-RNA vaccine construct, the ORF encoding the viral structural protein can be replaced with any chosen antigen, while the viral replicase remains an essential part of the vaccine, facilitating intracellular amplification of RNA after immunization.

[0338] Other activators In certain embodiments, the activators associated with the lipid particles of the present invention may comprise one or more therapeutic proteins, polypeptides, or small organic molecules or compounds. Non-limiting examples of such therapeutically effective agents or drugs include oncological agents (e.g., chemotherapeutic agents, hormone therapies, immunotherapies, radiotherapy agents, etc.), lipid-lowering agents, antiviral agents, anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, contraceptives, antipyretics, vasodilators, anti-angiogenic agents, cytovascular agents, signaling inhibitors, cardiovascular agents such as antiarrhythmics, hormones, vasoconstrictors, and steroids. These activators may be administered alone with the lipid particles of the present invention or in combination with (e.g., in combination with) the lipid particles of the present invention that contain nucleic acids such as interfering RNA or mRNA.

[0339] Non-exclusive examples of chemotherapy drugs include platinum-based drugs (e.g., oxaliplatin, cisplatin, carboplatin, spiroplatin, iproplatin, satraplatin, etc.), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechloretamine, uramustine, thiotepa, nitrosourea, etc.), antimetabolites (e.g., 5-fluorouracil (5-FU), azathioprine, methotrexate, leucovorin, capecitabine, cytarabine, phloxuridine, fludarabine, gemcitabine, pemetrexed, larcitrexed, etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel (Taxol), docetaxel, etc.), and topoisomers. Ace inhibitors (e.g., irinotecan (CPT-11, Camptosar), topotecan, amsacrin, etoposide (VP16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.), tyrosine kinases Inhibitors (e.g., gefitinib (Iressa®), sunitinib (Sutent®, SU11248), erlotinib (Tarceva®, OSI-1774), lapatinib (GW572016, GW2016), canertinib (CI1033), semaxinib (SU5416), batalanib (PTK787 / ZK222584), sorafenib (BAY) This includes 43-9006), imatinib (Gleevec®, STI571), dasatinib (BMS-354825), leflunomide (SU101), vandetanib (Zactima®, ZD6474), etc., pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogues thereof, and combinations thereof.

[0340] Examples of conventional hormone therapies include, but are not limited to, steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, tamoxifen, and goserelin, as well as other gonadotropin-releasing hormone agonists (GnRH).

[0341] Examples of conventional immunotherapies include immunostimulants (e.g., Calmette-Guérin bacillus (BCG), levamisole, interleukin-2, alpha-interferon, etc.), monoclonal antibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-pseudomonas exotoxin conjugate, etc.), and radioimmunotherapy (e.g., 111 In, 90 Y, or 131 This includes, but is not limited to, anti-CD20 monoclonal antibodies conjugated with I, etc.

[0342] Examples of conventional radiotherapy agents include those conjugated with antibodies targeting tumor antigens at the discretion of the patient. 47 Sc, 64 Cu, 67 Cu, 89 Sr, 86 Y, 87 Y, 90 Y, 105 Rh, 111 Ag, 111 In, 117m Sn, 149 PM, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 211 At, and 212 This includes, but is not limited to, radioactive nuclides such as Bi.

[0343] Further tumor agents that can be used according to the present invention include Alkeran, allopurinol, altretamine, amifostine, anastrozole, araC, arsenic trioxide, bexarotene, biCNU, carmustine, CCNU, celecoxib, cladribine, cyclosporine A, cytosine arabinoside, cytoxane, dexrazoxane, DTIC, estramustine, exemestane, FK506, gemtuzumab-ozogamicin, hydra, hydroxyurea, idarubicin, interferon, letrozole, and leustatin. Examples of oncological agents that can be used according to the present invention include, but are not limited to, leuprolide, litretinoin, megastrol, L-PAM, mesna, methoxsalen, mitramycin, nitrogen mustard, pamidronate, pegademase, pentostatin, porfimer sodium, prednisone, rituxan, streptozosin, STI-571, taxotere, temozolamide, VM-26, toremifene, tretinoin, ATRA, barrubicin, and vervan. Other examples of oncological agents that can be used according to the present invention include ellipticin and ellipticin analogs or derivatives, epotilon, intracellular kinase inhibitors, and camptothecin.

[0344] Non-exclusive examples of lipid-lowering agents for treating lipid disorders or conditions associated with elevated triglycerides, cholesterol, and / or glucose include statins, fibrates, ezetimibe, thiazolidinediones, niacin, beta-blockers, nitroglycerin, calcium antagonists, fish oil, and mixtures thereof.

[0345] Examples of antiviral drugs include abacavir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripra, cidofovir, combivir, darunavir, delavirdin, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfvirtide, entecavir, entry inhibitors, famciclovir, fixed-dose combination drugs, homivirsen, fosamprenavir, foscarnet, phosphonet, fusion inhibitors, ganciclovir, ivacitabine, immunovir, doxuridine, imiquimod, indinavir, inosine, integrase inhibitors, type III interferons (e.g., IFN- IFN-λ molecules such as λ1, IFN-λ2, and IFN-λ3, type II interferons (e.g., IFN-γ), type I interferons (e.g., IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ, such as PEGylated IFN-α), interferons, lamibucin, lopinavir, roviride, MK-0518, maraviroc, moloxidine, nelfinavir, nevirapine, nexavir, nucleoside analogs, oseltamivir, penciclovir, peramivir, preconalil, podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir, stabuzin, synergistic enhancers This includes, but is not limited to, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir, valganciclovir, bicribiloc, vidarabine, viramidine, zalcitabine, zanamivir, zidovudine, pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogues thereof, and mixtures thereof.

[0346] lipid particles The lipid particles of the present invention typically comprise an activator or therapeutic agent, a cationic lipid, a non-cationic lipid, and a conjugate lipid that inhibits particle aggregation. In some embodiments, the activator or therapeutic agent is completely encapsulated within the lipid portion of the lipid particle so that the activator or therapeutic agent in the lipid particle is resistant to enzymatic degradation by, for example, nucleases or proteases in aqueous solution. In other embodiments, the lipid particles described herein are substantially non-toxic to mammals such as humans. The lipid particles of the present invention typically have an average diameter of about 40 nm to about 150 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.

[0347] In preferred embodiments, the lipid particles of the present invention are serum-stable nucleic acid-lipid particles (LNPs) comprising one or more nucleic acid molecules such as interfering RNA (e.g., siRNA, aiRNA, and / or miRNA) or mRNA, cationic lipids (e.g., cationic lipids of formulas I, II, and / or III), non-cationic lipids (e.g., cholesterol alone or a mixture of one or more phospholipids and cholesterol), and conjugate lipids that inhibit particle aggregation (e.g., one or more PEG-lipid conjugates). The LNPs may contain at least one, two, three, four, five, six, seven, eight, nine, ten or more unmodified and / or modified nucleic acid molecules. Nucleic acid-lipid particles and methods for preparing them are described, for example, in U.S. Patents 5,753,613, 5,785,992, 5,705,385, 5,976,567, 5,981,501, 6,110,745, and 6,320,017, and PCT Publication WO96 / 40964, each of which is incorporated herein by reference in its entirety for all purposes.

[0348] Noncationic lipids 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.

[0349] Non-categorized lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidylcholine. This includes phatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dierydoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholines and diacylphosphatidylethanolamine phospholipids can also be used. The acyl group in these lipids is preferably 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.

[0350] 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.

[0351] 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.

[0352] 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.

[0353] In some embodiments, noncationic lipids may constitute about 13 mol% to about 49.5 mol%, about 20 mol% to about 45 mol%, about 25 mol% to about 45 mol%, about 30 mol% to about 45 mol%, about 35 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 40 mol%, or about 30 mol% to about 40 mol% of the total lipids present in the particles.

[0354] In certain embodiments, the cholesterol present in the phospholipid-free particles constitutes about 30 mol% to about 45 mol%, about 30 mol% to about 40 mol%, about 35 mol% to about 45 mol%, or about 35 mol% to about 40 mol% of the total lipids present in the particles. As a non-limiting example, phospholipid-free particles may contain cholesterol at about 37 mol% of the total lipids present in the particles.

[0355] In certain other embodiments, the cholesterol present in lipid particles containing a mixture of phospholipids and cholesterol constitutes about 30 mol% to about 40 mol%, about 30 mol% to about 35 mol%, or about 35 mol% to about 40 mol% of the total lipids present in the particles. As a non-limiting example, lipid particles containing a mixture of phospholipids and cholesterol may contain about 34 mol% cholesterol of the total lipids present in the particles.

[0356] In further embodiments, the cholesterol present in lipid particles containing a mixture of phospholipids and cholesterol constitutes about 10 mol% to about 30 mol%, about 15 mol% to about 25 mol%, or about 17 mol% to about 23 mol% of the total lipids present in the particles. As a non-limiting example, lipid particles containing a mixture of phospholipids and cholesterol may contain about 20 mol% cholesterol of the total lipids present in the particles.

[0357] In embodiments in which lipid particles contain a mixture of phospholipids and cholesterol or cholesterol derivatives, the mixture may constitute up to about 40, 45, 50, 55, or 60 mol% of the total lipids present in the particles. In certain cases, the phospholipid component in the mixture may constitute about 2 mol% to about 12 mol%, about 4 mol% to about 10 mol%, about 5 mol% to about 10 mol%, about 5 mol% to about 9 mol%, or about 6 mol% to about 8 mol% of the total lipids present in the particles. As a non-limiting example, lipid particles containing a mixture of phospholipids and cholesterol may contain about 7 mol% of the total lipids present in the particles, such as phospholipids like DPPC or DSPC (for example, in a mixture with about 34 mol% cholesterol). In certain other cases, the phospholipid component in the mixture may constitute about 10 mol% to about 30 mol%, about 15 mol% to about 25 mol%, or about 17 mol% to about 23 mol% of the total lipids present in the particles. As another non-limiting example, lipid particles containing a mixture of phospholipids and cholesterol may contain phospholipids such as DPPC or DSPC (for example, in a mixture with about 20 mol% cholesterol) of the total lipids present in the particles.

[0358] Lipid conjugate In addition to cationic and non-cationic lipids, the lipid particles of the present invention (e.g., LNPs) include lipid conjugates. Conjugated lipids are useful in inhibiting particle aggregation. Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, ATTA-lipid conjugates, cationic polymer-lipid conjugates (CPLs), and mixtures thereof. In certain embodiments, the particles include either PEG-lipid conjugates or ATTA-lipid conjugates together with CPLs.

[0359] In preferred embodiments, the lipid conjugate is a PEG-lipid. Examples of PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyl (PEG-DAA), as described in PCT Publication 05 / 026372; PEG coupled to diacylglycerol (PEG-DAG), as described in U.S. Patent Application Publications 20030077829 and 2005008689; PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE); PEG conjugated to ceramide, as described in U.S. Patent No. 5,885,613; PEG conjugated to cholesterol or its derivatives; and mixtures thereof. The disclosures of these patent documents are incorporated herein by reference in their entirety for all purposes. Further PEG-lipids include, but are not limited to, PEG-C-DOMG, 2KPEG-DMG, and mixtures thereof.

[0360] PEG is a linear, water-soluble polymer of ethylene PEG repeating units having two terminal hydroxyl groups. PEG is classified by its molecular weight; for example, PEG2000 has an average molecular weight of approximately 2,000 daltons, and PEG5000 has an average molecular weight of approximately 5,000 daltons. PEG is commercially available from Sigma Chemical Co. and other companies and includes, for example, monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol succinate (MePEG-S), monomethoxypolyethylene glycol succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol amine (MePEG-NH2), monomethoxypolyethylene glycol toresylate (MePEG-TRES), and monomethoxypolyethylene glycol imidazolyl carbonyl (MePEG-IM). Other PEGs (e.g., mPEG(20kDa)amines), such as those described in U.S. Patents 6,774,180 and 7,053,150, are also useful in the preparation of the PEG-lipid conjugates of the present invention. The disclosures of these patents are incorporated herein by reference in their entirety for all purposes. Furthermore, monomethoxypolyethylene glycol acetate (MePEG-CH2COOH) is particularly useful in the preparation of PEG-lipid conjugates, including PEG-DAA conjugates, for example.

[0361] The PEG portion of the PEG-lipid conjugates described herein may have an average molecular weight in the range of about 550 daltons to about 10,000 daltons. In specific cases, the PEG portion has an average molecular weight of about 750 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG portion has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0362] In certain cases, PEG may be optionally substituted with alkyl groups, alkoxy groups, acyl groups, or aryl groups. PEG may be directly conjugated to lipids or linked to lipids via a linker moiety. For example, any linker moiety suitable for coupling PEG to lipids can be used, including ester-free and ester-containing linker moieties. In preferred embodiments, the linker moiety is an ester-free linker moiety. As used herein, the term “ester-free linker moiety” refers to a linker moiety that does not contain a carboxylic acid ester bond (-OC(O)-). Suitable ester-free linker moieties include, but are not limited to, amides (-C(O)NH-), aminos (-NR-), carbonyls (-C(O)-), carbamates (-NHC(O)O-), ureas (-NHC(O)NH-), disulfides (-SS-), ethers (-O-), succinyls (-(O)CCH2CH2C(O)-), succinamidyls (-NHC(O)CH2CH2C(O)NH-), ethers, disulfides, and combinations thereof (e.g., linkers containing both carbamate and amide linker moieties). In preferred embodiments, carbamate linkers are used to couple PEG to lipids.

[0363] In other embodiments, the ester-containing linker moiety is used to couple PEG to lipids. Suitable ester-containing linker moieties include, for example, carbonates (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonic acid esters, and combinations thereof.

[0364] Further PEG-lipid conjugates suitable for use in the present invention include, but are not limited to, formula: [ka] [In the formula, A is (C1-C6) alkyl, (C3-C8) cycloalkyl, (C3-C8) cycloalkyl, (C1-C6) alkyl, (C1-C6) alkoxy, (C2-C6) alkenyl, (C2-C6) alkynyl, (C1-C6) alkanoyl, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, or (C2-C6) alkanoyloxy, where any (C1-C6) alkyl, (C3-C8) cycloalkyl, (C3-C8) cycloalkyl, (C1-C6) alkyl, (C1-C6) alkoxy, (C2-C6) alkenyl, (C2-C6) alkynyl, (C1-C6) alkanoyl, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, and (C2-C6) alkanoyl The yloxy is substituted with one or more anionic precursor groups, where any (C1-C6) alkyl, (C3-C8) cycloalkyl, (C3-C8) cycloalkyl(C1-C6) alkyl, (C1-C6) alkoxy, (C2-C6) alkenyl, (C2-C6) alkynyl, (C1-C6) alkanoyl, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, and (C2-C6) alkanoyloxy is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, (C1-C3) alkoxy, (C1-C6) alkanoyl, (C1-C3) alkoxycarbonyl, (C1-C3) alkylthio, or (C2-C3) alkanoyloxy; B is a polyethylene glycol chain having a molecular weight of approximately 550 daltons to approximately 10,000 daltons; C is -LR a (L represents direct bond, -C(O)O-, -C(O)NR) b -, -NR b -, -C(O)-, -NR b C(O)O-, -NR b C(O)NR b Selected from the group consisting of -, -SS-, -O-, -(O)CCH2CH2C(O)-, and -NHC(O)CH2CH2C(O)NH-; R a is branched (C 10 ~C 50) Alkyl or branched (C 10 ~C 50 ) is an alkenyl, and here, branched (C 10 ~C 50 ) Alkyl or branched (C 10 ~C 50 ) One or more carbon atoms of the alkenyl are substituted with -O-; R b Each of these compounds is independently H or (C1-C6) alkyl, or a salt thereof.

[0365] Conjugate lipids may include, for example, PEG-lipids comprising compounds of the formula A-PEG-diacylglycerol (DAG), A-PEG-dialkyloxypropyl (DAA), A-PEG-phospholipid, A-PEG-ceramide (Cer), or mixtures thereof, where A is (C1-C6)alkyl, (C3-C8)cycloalkyl, (C3-C8)cycloalkyl(C1-C6)alkyl, (C1-C6)alkoxy, (C2 ~C6) alkenyl, (C2~C6) alkynyl, (C1~C6) alkanoyl, (C1~C6) alkoxycarbonyl, (C1~C6) alkylthio, or (C2~C6) alkanoyloxy, where any (C1~C6) alkyl, (C3~C8) cycloalkyl, (C3~C8) cycloalkyl, (C1~C6) alkyl, (C1~C6) alkoxy, (C2~C6) alkenyl, (C2~C6) alkynyl, (C1-C6) alkanoyl, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, and (C2-C6) alkanoyloxy are substituted with one or more anionic precursor groups, wherein any (C1-C6) alkyl, (C3-C8) cycloalkyl, (C3-C8) cycloalkyl(C1-C6) alkyl, (C1-C6) alkoxy, (C2-C6) alkenyl, (C2-C6) alkynyl, ( C1-C6) alkanoyl, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, and (C2-C6) alkanoyloxy are optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, (C1-C3) alkoxy, (C1-C6) alkanoyl, (C1-C3) alkoxycarbonyl, (C1-C3) alkylthio, or (C2-C3) alkanoyloxy. The A-PEG-DAA conjugate may be A-PEG-dilauryloxypropyl (C12), A-PEG-dimyristyloxypropyl (C14), A-PEG-dipalmityloxypropyl (C16), or A-PEG-distearyloxypropyl (C18), or a mixture thereof.

[0366] Lipid conjugates can be formed by conjugating PEG with phosphatidylethanolamines having various acyl chain groups with different chain lengths and saturations. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. 10 ~C 20 Phosphatidylethanolamines containing saturated or unsaturated fatty acids having carbon chain lengths in the range of are preferred. Phosphatidylethanolamines containing mono- or diunsaturated fatty acids, and mixtures of saturated and unsaturated fatty acids, can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0367] The terms “ATTR” or “polyamide” refer, not limited to, to the compounds described in U.S. Patent Nos. 6,320,017 and 6,586,559 (these disclosures are incorporated herein by reference in their entirety for all purposes). These compounds include those with the formula: [ka] (wherein R is a member selected from the group consisting of hydrogen, alkyl and acyl; R 1 is a member selected from the group consisting of hydrogen and alkyl; or optionally, R and R 1 Furthermore, the nitrogen atoms to which they bind form an azide moiety; R 2 R is a member of the group selected from hydrogen, optionally substituted alkyl, optionally substituted aryl, and amino acid side chains; 3 These include hydrogen, halogen, hydroxyl, alkoxyl, mercapto, hydrazino, amino, and NR 4 R 5 (In the formula, R 4 and R5 The compounds include a member selected from the group consisting of (which is independently hydrogen or alkyl); n is 4 to 80; m is 2 to 6; p is 1 to 4; and q is 0 or 1. It will be apparent to those skilled in the art that other polyamides can be used in the compounds of the present invention.

[0368] The term "diacylglycerol" refers to two fatty acid acyl chains, R 1 and R 2 This refers to compounds having the following characteristics, both of which independently have 2 to 30 carbon atoms bonded to the 1st and 2nd positions of glycerol by ester bonds. The acyl group may be saturated or have various degrees of unsaturation. A suitable acyl group is lauryl(C) 12 ), Millistil (C 14 ), Palmityl (C 16 ), Stearyl (C 18 ), and Icosil (C 20 This includes, but is not limited to, ) and R. In a preferred embodiment, 1 and R 2 They are the same, for example, R 1 and R 2 These are all myristyls (for example, dimyristyl), R 1 and R 2 These are all stearyl compounds (e.g., distearyl), etc. Diacylglycerol has the following general formula: [ka] It has.

[0369] The term "dialkyloxypropyl" refers to two alkyl chains, R 1 and R 2 This refers to compounds having the following characteristics, both of which independently have 2 to 30 carbon atoms. The alkyl group may be saturated or have varying degrees of unsaturation. Dialkyloxypropyl is defined by the following general formula: [ka] It has.

[0370] In a preferred embodiment, the PEG-lipid is given by the following formula: [ka] It is a PEG-DAA conjugate having (wherein R 1 and R 2 ( is independently selected and is a long-chain alkyl group having about 10 to about 22 carbon atoms; PEG is polyethylene glycol; L is the aforementioned ester-free or ester-containing linker moiety). The long-chain alkyl group can be saturated or unsaturated. Suitable alkyl groups include lauryl (C 12 ), Millistil (C 14 ), Palmityl (C 16 ), Stearyl (C 18 ), and Icosil (C 20 This includes, but is not limited to, ) and R. In a preferred embodiment, 1 and R 2 They are the same, for example, R 1 and R 2 These are all myristyls (for example, dimyristyl), R 1 and R 2 These are all stearyl compounds (for example, distearyl), and so on.

[0371] In formula VII above, PEG has an average molecular weight in the range of about 550 daltons to about 10,000 daltons. In specific cases, PEG has an average molecular weight of about 500 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, PEG has an average molecular weight of about 2,000 daltons or about 750 daltons. PEG may optionally be substituted with alkyl, alkoxy, acyl, or aryl groups. In specific embodiments, terminal hydroxyl groups are substituted with methoxy or methyl groups.

[0372] In a preferred embodiment, "L" is an ester-free linker portion. Preferred ester-free linkers include, but are not limited to, amide linker portions, amino linker portions, carbonyl linker portions, carbamate linker portions, urea linker portions, ether linker portions, disulfide linker portions, succinamidyl linker portions, and combinations thereof. In a preferred embodiment, the ester-free linker portion is a carbamate linker portion (e.g., PEG-C-DAA conjugate). In another preferred embodiment, the ester-free linker portion is an amide linker portion (e.g., PEG-A-DAA conjugate). In yet another preferred embodiment, the ester-free linker portion is a succinamidyl linker portion (e.g., PEG-S-DAA conjugate).

[0373] In certain embodiments, the PEG-lipid conjugate is selected from the following: [ka] In one embodiment, n is selected such that the resulting polymer chain has a molecular weight of about 2000.

[0374] PEG-DAA conjugates are synthesized using standard techniques and reagents known to those skilled in the art. It will be recognized that PEG-DAA conjugates contain various amide, amine, ether, thio, carbamate, and urea bonds. Those skilled in the art will recognize that methods and reagents for forming these bonds are well known and readily available. See, for example, March, Advanced Organic Chemistry (Wiley 1992), Larock, Comprehensive Organic Transformations (VCH 1989), and Furniss, Voguel's Textbook of Practical Organic Chemistry, 5th ed. (Longman 1989). It will also be understood that any functional groups present may require protection and deprotection at various points in the synthesis of PEG-DAA conjugates. Those skilled in the art will recognize that such techniques are well known. For example, see Green and Wuts, Protective Groups in Organic Synthesis (Wiley 1991).

[0375] Preferably, the PEG-DAA conjugate is dilauryloxypropyl (C 12 )-PEG conjugate, dimyristyloxypropyl (C 14 )-PEG conjugate, dipalmityloxypropyl (C 16 )-PEG conjugate, or distearyloxypropyl (C 18 It is a PEG conjugate. Those skilled in the art will readily understand that other dialkyloxypropyls can be used in the PEG-DAA conjugate of the present invention.

[0376] In addition to the foregoing, it will be readily apparent to those skilled in the art that other hydrophilic polymers can be used instead of PEG. Examples of suitable polymers that can be used instead of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized cellulose such as hydroxymethylcellulose or hydroxyethylcellulose.

[0377] The charge of the polycationic moiety may be distributed throughout the entire particle moiety, or it may be a charge density of different concentrations in a particular region of the particle moiety, such as a charge spike. If the charge density is distributed on the particle, it may be uniformly or non-uniformly distributed. All variations of the charge distribution of the polycationic moiety are encompassed by the present invention.

[0378] Lipid "A" and non-immunogenic polymer "W" can be bonded together by various methods, preferably by covalent bonds. Methods known to those skilled in the art can be used for covalent bonding between "A" and "W". Suitable bonds include, but are not limited to, amide bonds, amine bonds, carboxyl bonds, carbonate bonds, carbamate bonds, ester bonds, and hydrazone bonds. It will be obvious to those skilled in the art that "A" and "W" must have complementary functional groups to result in a bond. The desired bond is obtained by the reaction of these two groups, one on the lipid and the other on the polymer. For example, if the lipid is diacylglycerol and its terminal hydroxyl is activated, for example, with NHS and DCC to form an active ester, and then this lipid reacts with a polymer containing amino groups, such as a polyamide (see, for example, U.S. Patents 6,320,017 and 6,586,559; their disclosures are incorporated herein by reference in their entirety for all purposes), an amide bond is formed between the two groups.

[0379] In certain cases, the polycationic moiety may be bound to a ligand such as a targeted ligand or a chelate moiety for complexing calcium. Preferably, the cationic moiety maintains a positive charge after ligand binding. In certain cases, the bound ligand has a positive charge. Suitable ligands include, but are not limited to, compounds or devices having reactive functional groups, and include lipids, amphiphilic lipids, carrier compounds, biocompatible compounds, biomaterials, biopolymers, medical devices, analytically detectable compounds, therapeutically active compounds, enzymes, peptides, proteins, antibodies, immunostimulants, radiolabels, fluorescent agents, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, visomes, micelles, immunoglobulins, functional groups, other targeted moieties, or toxins.

[0380] Lipid conjugates (e.g., PEG-lipids) typically constitute about 0.1 mol% to 10 mol%, 0.5 mol% to 10 mol%, 1 mol% to 10 mol%, 0.6 mol% to 1.9 mol%, 0.7 mol% to 1.8 mol%, 0.8 mol% to 1.7 mol%, 0.9 mol% to 1.6 mol%, 0.9 mol% to 1.8 mol%, 1 mol% to 1.8 mol%, 1 mol% to 1.7 mol%, 1.2 mol% to 1.8 mol%, 1.2 mol% to 1.7 mol%, 1.3 mol% to 1.6 mol%, or 1.4 mol% to 1.5 mol% of the total lipids present in the particles.

[0381] Those skilled in the art will understand that the concentration of the lipid conjugate can be varied depending on the lipid conjugate used and the rate at which the nucleic acid-lipid particles become membrane-fused.

[0382] By controlling the composition and concentration of the lipid conjugate, the rate at which the lipid conjugate replaces the outside of the nucleic acid-lipid particles, and subsequently the rate at which the nucleic acid-lipid particles become membrane-fusing, can be controlled. For example, when using a PEG-phosphatidylethanolamine conjugate or a PEG-ceramide conjugate as the lipid conjugate, the rate at which the nucleic acid-lipid particles become membrane-fusing can be altered, for example, by changing the concentration of the lipid conjugate, by changing the molecular weight of PEG, or by changing the chain length and saturation of the acyl chain group on the phosphatidylethanolamine or ceramide. Furthermore, the rate at which the nucleic acid-lipid particles become membrane-fusing can be altered and / or controlled using other variables, such as pH, temperature, and ionic strength. Other methods that can be used to control the rate at which nucleic acid-lipid particles become membrane-fusing will become apparent to those skilled in the art by reading this disclosure.

[0383] Preparation of lipid particles Lipid particles of the present invention, such as LNPs, in which an active or therapeutic agent, such as a nucleic acid molecule, is encapsulated in a lipid bilayer and protected from degradation, can be formed by any method known in the art, including but not limited to a continuous mixing method or a direct dilution process.

[0384] In preferred embodiments, the cationic lipid is a lipid of formulas I, II, and III, or a combination thereof. In other preferred embodiments, the non-cationic lipid is egg sphingomyelin (ESM), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dipalmitoylphosphatidylcholine (DPPC), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, 14:0 PE (1,2-dimyristoylphosphatidylethanolamine (DMPE)), 16:0 PE (1,2-dipalmitoylphosphatidylethanolamine (DPPE)), 18:0 PE (1,2-distearoylphosphatidylethanolamine (DSPE)), 18:1 PE(1,2-dioleoyl-phosphatidylethanolamine (DOPE)), 18:1 transPE(1,2-dierydoyl-phosphatidylethanolamine (DEPE)), 18:0-18:1 PE(1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE)), 16:0-18:1 PE(1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)), polyethylene glycol polymers (e.g., PEG2000, PEG5000, PEG-modified diacylglycerol, or PEG-modified dialkyloxypropyl), cholesterol, or combinations thereof.

[0385] In certain embodiments, the present invention provides LNPs produced by a continuous mixing method, for example, a process comprising supplying an aqueous solution containing a nucleic acid such as interfering RNA or mRNA 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 acids (e.g., interfering RNA or mRNA). The process and apparatus for carrying out the process are described in detail in U.S. Patent Application Publication No. 20040142025, which is incorporated herein by reference in its entirety for all purposes.

[0386] The continuous introduction of lipids and a buffer solution into a mixing environment, such as a mixing chamber, results in serial dilution of the lipid solution by the buffer solution, 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 (e.g., an aqueous solution) to generate nucleic acid-lipid particles.

[0387] LNPs formed using the continuous mixing method typically have sizes of approximately 40 nm to 150 nm, 50 nm to 150 nm, 60 nm to 130 nm, 70 nm to 110 nm, or 70 nm to 90 nm. The particles formed in this way do not aggregate and are optionally sized to obtain a uniform particle size.

[0388] 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, advantageously, yield smaller particles if introduced into a collection container containing an equal volume of dilution buffer.

[0389] 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 is advantageous in that it allows for further control of the flow of the dilution buffer mixing with the liposome solution in the second mixing region, and therefore also of the concentration of the liposome solution in the buffer throughout the second mixing process. By controlling the flow rate of the dilution buffer in this way, it becomes advantageous to be able to form small particles at reduced concentrations.

[0390] The apparatus for carrying out these processes and these direct dilution processes is described in detail in U.S. Patent Application Publication No. 20070042031, which is incorporated herein by reference in its entirety for all purposes.

[0391] LNPs formed using the direct dilution process typically have sizes ranging from approximately 40 nm to 150 nm, 50 nm to 150 nm, 60 nm to 130 nm, 70 nm to 110 nm, or 70 nm to 90 nm. The resulting particles do not aggregate and are optionally sized to obtain a uniform particle size.

[0392] If necessary, the lipid particles of the present invention (e.g., LNPs) can be sized by any method available for liposome sizing. Sizing can be performed to obtain a desired size range and a relatively narrow particle size distribution.

[0393] 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, 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 to about 80 nm) is observed. In both methods, the particle size distribution can be monitored by conventional laser particle size identification or QELS.

[0394] 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 clear size distribution. Typically, the suspension is circulated through the membrane one or more times until the desired particle size distribution is obtained. The size can be gradually reduced by sequentially passing the particles through membranes with smaller pores and extruding them.

[0395] 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).

[0396] 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.

[0397] In some embodiments, the nucleic acid-to-lipid ratio (mass / mass ratio) in the formed LNPs is in the range of about 0.01 to about 0.2, about 0.02 to about 0.1, about 0.03 to about 0.1, or about 0.01 to about 0.08. The ratio of the starting materials also falls within this range. In other embodiments, the LNP preparation uses about 400 μg of nucleic acid per 10 mg of total lipid, or a nucleic acid-to-lipid mass ratio of about 0.04, more preferably corresponding to 1.25 mg of total lipid per 50 μg of nucleic acid. In other preferred embodiments, the particles have a nucleic acid:lipid mass ratio of about 0.08.

[0398] In other embodiments, the lipid-to-nucleic acid ratio (mass / mass ratio) in the formed LNP is approximately 1 (1:1) to approximately 100 (100:1), approximately 5 (5:1) to approximately 100 (100:1), approximately 1 (1:1) to approximately 50 (50:1), approximately 2 (2:1) to approximately 50 (50:1), approximately 3 (3:1) to approximately 50 (50:1), and approximately 4 (4:1) )~approximately 50 (50:1), approximately 5 (5:1)~approximately 50 (50:1), approximately 1 (1:1)~approximately 25 (25:1), approximately 2 (2:1)~approximately 25 (25:1), approximately 3 (3:1)~approximately 25 (25:1), approximately 4 (4:1)~approximately 25 (25:1), approximately 5 (5:1)~approximately 25 (25:1), approximately 5 (5:1)~approximately 20 (20: 1) Approximately 5 (5:1) to approximately 15 (15:1), approximately 5 (5:1) to approximately 10 (10:1), approximately 5 (5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9 (9:1), (10:1), 11 (11:1), 12 (12:1), 13 (13:1), 14 (14:1), 15 (15:1), 16 (16:1) The ratios are in the range of 17 (17:1), 18 (18:1), 19 (19:1), 20 (20:1), 21 (21:1), 22 (22:1), 23 (23:1), 24 (24:1), 25 (25:1), 26 (26:1), 27 (27:1), 28 (28:1), 29 (29:1), or 30 (30:1). The ratios of the starting materials also usually fall within this range.

[0399] As mentioned above, conjugated lipids may further contain CPLs. Various general methods for preparing LNP-CPLs (CPL-containing LNPs) are discussed herein. Two general techniques include the “post-insertion” technique, i.e., insertion of CPLs into pre-formed LNPs, and the “standard” technique, i.e., incorporating CPLs into the lipid mixture during the LNP formation process. The post-insertion technique produces LNPs with CPLs mainly on the outer surface of the LNP bilayer membrane, while the standard technique yields LNPs with CPLs on both the inner and outer surfaces. This method is particularly useful for vesicles made from phospholipids (which may contain cholesterol), and also for vesicles containing PEG-lipids (e.g., PEG-DAA and PEG-DAG). Methods for producing LNP-CPLs are taught, for example, in U.S. Patents 5,705,385, 6,586,410, 5,981,501, 6,534,484, and 6,852,334, U.S. Patent Application Publication 20020072121, and PCT Publication 00 / 62813, and these disclosures are incorporated herein by reference in their entirety for all purposes.

[0400] Other methods for producing LNPs can be found, for example, in U.S. Patent No. 9,005,654 and PCT Publication No. 2007 / 012191, and these disclosures are incorporated herein by reference in their entirety for all purposes.

[0401] kit The present invention also provides lipid particles (e.g., LNPs) in kit form. The kit may include a compartmentalized container to hold various components of the lipid particles (e.g., activators or therapeutic agents such as nucleic acids, and individual lipid components of the particles). In some embodiments, the kit may further include an endosomal membrane destabilizer (e.g., calcium ions). The kit typically contains the lipid particle composition of the present invention, preferably in a dehydrated form, along with instructions for its rehydration and administration.

[0402] As described herein, the lipid particles (e.g., LNPs) of the present invention can be adapted to preferentially target specific tissues, organs, or tumors of interest. In certain cases, preferential targeting of lipid particles such as LNPs can be achieved by controlling the composition of the particles themselves. For example, as described in Example 11, a 1:57 PEG-cDSA LNP formulation can be used to preferentially target tumors outside the liver, while a 1:57 PEG-cDMA LNP formulation has been found to preferentially target the liver (including hepatomas).

[0403] In certain other cases, it may be desirable to have a targeting moiety bound to the surface of the lipid particle in order to further improve particle targeting. Methods for binding a targeting moiety (e.g., an antibody, a protein, etc.) to a lipid (such as the one used in the particles of this invention) are known to those skilled in the art.

[0404] Administration of lipid particles The lipid particles (e.g., LNPs) of the present invention are useful for introducing activators or therapeutic agents (e.g., nucleic acids such as interfering RNA or mRNA) into cells after they have been formed. Accordingly, the present invention also provides a method for introducing activators or therapeutic agents, such as 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 the activator or therapeutic agent to the cells to occur.

[0405] 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 active or therapeutic (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 particle contents mix with the intracellular fluid.

[0406] 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. Lipids may be frozen for stabilization. For example, lipids can be stored at -20°C in a pH 8 Tris buffer with a high salt concentration (e.g., 500 mM NaCl). In a further example, lipids can also be stored at -80°C in a mixture containing sucrose and maltose in a pH 8 Tris buffer. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, etc., and glycoproteins for enhanced stability, such as albumin, lipoproteins, globulins, etc. Further preferred 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 retardants, buffers, carrier solutions, suspensions, colloids, etc. The term “pharmaceutically acceptable” means molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans.

[0407] 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.

[0408] The concentration of particles in a pharmaceutical formulation can vary considerably, from, for example, less than about 0.05% by weight, typically around 2–5% by weight or at least about 2–5% by weight, to as high as 10–90% by weight, and is selected according to the chosen specific mode of administration, primarily based on fluid volume, viscosity, etc. For example, the concentration can 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.

[0409] 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 and buffers, osmotic pressure adjusters, etc., 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.

[0410] 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, as described in PCT Publications WO05 / 007196, WO05 / 121348, WO05 / 120152, and WO04 / 002453 (these disclosures are incorporated herein by reference in their entirety for all purposes). 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.

[0411] 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, for example, 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 aforementioned references are incorporated herein by reference in their entirety for all purposes.

[0412] 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) (for example, 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.

[0413] In certain embodiments, pharmaceutical compositions can 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.

[0414] 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.

[0415] Generally, for intravenous administration, lipid particle formulations are formulated using a suitable pharmaceutical carrier. Many pharmaceutically acceptable carriers can be used in the compositions and methods of the present invention. Formulations suitable 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 can be used, such as water, buffered water, 0.4% saline, 0.3% glycine, etc., and these aqueous carriers may contain glycoproteins for stability enhancement, such as albumin, lipoproteins, and globulins. Generally, buffered saline (135-150 mM NaCl) is used as a pharmaceutically acceptable carrier, but other suitable carriers may 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 may be sterilized using the techniques described above, or alternatively, they may be produced under sterile conditions. The resulting aqueous solutions may be packaged for use, or filtered and freeze-dried under sterile conditions, and the freeze-dried preparation may be mixed with the sterile aqueous solution before administration.

[0416] 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 all purposes). These oral dosage forms may also contain, namely, binders, gelatin; excipients, lubricants, and / or flavoring agents. If the unit dosage form is a capsule, the dosage form may contain a liquid carrier in addition to the aforementioned substances. Various other substances may be present as coating agents 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.

[0417] Typically, these oral formulations may contain at least about 0.1% or more of 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 can 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.

[0418] Formulations suitable for oral administration may consist of: (a) an effective amount of a packaged therapeutic agent, 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 one or more of the following: lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and 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, such as a fragrance, a therapeutic agent such as nucleic acid (e.g., interfering RNA or mRNA) in sucrose, and a carrier known in the art in addition to the therapeutic agent.

[0419] 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.

[0420] 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.

[0421] 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.

[0422] 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, as well as 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.

[0423] 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.

[0424] When performed in vitro, contact between cells and lipid particles takes place in a biocompatible culture medium. The particle concentration varies widely depending on the specific application, but is generally between approximately 1 μmol and 10 mmol. Treatment of cells with lipid particles is generally carried out at physiological temperature (approximately 37°C) for a period of approximately 1 to 48 hours, preferably approximately 2 to 4 hours.

[0425] 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.

[0426] 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 Application Publication No. 20030077829, 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.). 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.

[0427] 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 preferred embodiments, an activator or therapeutic agent, such as one or more nucleic acid molecules (e.g., interfering RNA (e.g., siRNA) or mRNA), is 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, appendiceal 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.

[0428] 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).

[0429] To the extent necessary, cell tissue culture is well known in the 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 guidance for cell culture. Cultured cell systems are often in the form of monolayer cells, but cell suspensions are also used.

[0430] 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) or mRNA sequences, by detection of target sequences (e.g., by detecting the expression or reduction of expression of the target sequence), or by a combination thereof.

[0431] 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 These include, but are 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.

[0432] Nucleic acid detection Nucleic acids (e.g., interfering RNA or mRNA) are detected and quantified by any means well known to those skilled in the art as described herein. Detection of nucleic acids can be carried out by well known methods such as Southern spectroscopy, Northern spectroscopy, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. Further analytical biochemical methods such as spectrophotography, radiography, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and hyperdiffusion chromatography can also be used.

[0433] 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).

[0434] The sensitivity of hybridization assays can be improved through the use of nucleic acid amplification systems that increase the amount of the target nucleic acid detected. In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for preparing nucleic acid fragments for subsequent subcloning are known.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 cited in 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 nucleic acids amplified 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 mutants 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, nonspecific PCR primers, and then the amplified target region can be searched for for specific sequences exhibiting mutations. The disclosures of the aforementioned references are incorporated herein by reference in their entirety for all purposes.

[0435] 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 automated synthesizers, such as those described in 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 in 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.

[0436] 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]

[0437] 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 invention in any way. Those skilled in the art will readily recognize various non-essential parameters that can be changed or modified to produce essentially the same results.

[0438] Example 1 Synthesis of (4-bromobutyl)tris[(4Z)-deca-4-en-1-yloxy]silane (3) [ka] (4-bromobutyl)trichlorosilane (1) (21.77 g, 80.5 mmol) was stirred in Et2O at 0°C. While stirring, Et2O containing (4Z)-deca-4-en-1-ol (2) (41.5 g, 265.6 mmol) and TEA (36.9 mL, 265.6 mmol) was added dropwise. After the addition was complete, the flask was stopped, shaken thoroughly, and allowed to stand overnight at room temperature. The resulting precipitate was removed by filtration, and the cake was washed with additional Et2O. The filtrate was concentrated under reduced pressure, and the residue was purified by automated flash purification (1% siRNA / Hex) to obtain (4-bromobutyl)tris[(4Z)-deca-4-en-1-yloxy]silane (36.18 g, 71.4%). 1H NMR(400MHz,CDCl3)δ5.38(m,6H),3.74(t,6H,J=4Hz),2.22(m 8H), 2-2.12(m, 12H), 1.61(m, 6H), 1.24-1.52(m, 28H), 0.89(t, 9H, J=4Hz), J=0.64(m, 2H).

[0439] Example 2 Synthesis of Compounds 4 and 5 [ka] Compounds (4 and 5) were synthesized using appropriate alcohols in the same manner as (4-bromobutyl)tris[(4Z)-deca-4-en-1-yloxy]silane (3).

[0440] Example 3 Synthesis of (4-bromobutyl)bis(((Z)-deca-4-en-1-yl)oxy)(methyl)silane (7) [ka] Compound (7) was prepared using (4-bromobutyl)dichloro(methyl)silane (6) in the same manner as compound (3). (9.5 g, 51%) was obtained. 1 H NMR(400MHz,CDCl3)δ5.36(m,4H),3.68(4H,t,J=8Hz),3.41(t,2H,J=8Hz),1.99-2.11( m,8H),1.49-1.64(m,6H),1.31(m,12H),0.89(t,6H,J=4Hz),0.61(m,2H),0.11(s,3H).

[0441] Example 4 Synthesis of compounds 8, 9, and 10. [ka] Compounds (8, 9, and 10) were synthesized using appropriate alcohols in the same manner as (4-bromobutyl)bis(((Z)-deca-4-en-1-yl)oxy)(methyl)silane (7).

[0442] Example 5: The general synthesis of compounds 13-43 in Table 1 is illustrated by the synthesis of compound 12. [ka] Synthesis of N1,N3-bis(4-(bis(((Z)-deca-4-en-1-yl)oxy)(methyl)silyl)butyl)-N1,N3-dimethylpropane-1,3-diamine(12): (4-bromobutyl)bis(((Z)-deca-4-en-1-yl)oxy)(methyl)silane(7) (0.72 g, 1.47 mmol), N1,N3-dimethylpropane-1,3-diamine(11) (50 mg, 0.49 mmol), and K2CO3 (138 mg, 1.22 mmol) were heated in a microwave at 150°C for 25 minutes. The reaction mixture was diluted with SiO2, washed with H2O and saturated NaCl solution, separated, dried, and concentrated under reduced pressure in (MgSO4). The residue was purified by automated flash chromatography (5% MeOH / DCM) to obtain N1,N3-bis(4-(bis(((Z)-deca-4-en-1-yl)oxy)(methyl)silyl)butyl)-N1,N3-dimethylpropane-1,3-diamine(12) (96 mg, 21.3%). 1 H NMR(400MHz,CDCl3)δ5.38(m,8H),3.66(t,8H,J=4Hz),2.8(m,2H),2.68(m,2H),2.49(s,6H ),(1.98-2.11)(m,18H),1.56-1.72(m,30H),0.88(t,12H,J=4Hz),0.62(m,4H),0.1(s,6H).

[0443] The compounds (13-43) in Table 1 were synthesized using appropriate diamine and silicon halide starting materials, employing a similar synthetic methodology. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13]

[0444] Therefore, a particular embodiment of the present invention relates to any one of the compounds described herein, including any one of compounds 12 to 43, and a salt thereof.

[0445] Example 6 General formulation procedure The lipid solution contained four components: PEG-conjugated lipids, ionizing lipids (e.g., silicon lipids), cholesterol, and phospholipids (e.g., DSPC). The lipid stock solution was prepared using the described lipid identities and molar ratios. siRNA was diluted in 100 mM acetate buffer at pH 4 to prepare lipid nanoparticles (LNPs) with a total lipid to siRNA weight ratio of approximately 10:1–20:1. Equivolutes of lipid solution and nucleic acid solution were mixed through a T-connector at a flow rate of 400 mL / min and diluted with pH 7.4 PBS. Ethanol was then removed, and the outer buffer was replaced with Tris / NaCl buffer by dialysis. After dialysis, the formulation was concentrated using a VivaSpin concentrator unit (MWCO 100,000) and filtered through a sterile filter with a pore size of 0.2 μm. Nucleic acid concentration was determined by the RiboGreen assay. Particle size and polydispersity were determined using a Malvern Nano Series Zetasizer.

[0446] Activity evaluation Generally, HSC-LNP activity was measured by intravenous injection of LNP preparations at a dose of 0.025 mg / kg into female Balb / C mice (5-8 weeks old). Immediately before injection, the siRNA LNP stock solution was filtered and diluted to the required dosage concentration. 48 hours after administration (the final time point), the animals were euthanized with a lethal dose of ketamine / xylazine. Half of the left lobe of the liver was collected in 1.5 mL of RNALater and stored overnight at 2-8°C. The following day, approximately 20-25 mg of liver tissue was homogenized, and the liver lysates were used in the QuantiGene assay to examine the relative levels of target mRNA and GAPDH expression in mice. Data from each animal were normalized to the liver weight used in the assay, and then normalized relative to the GAPDH signal; data are reported as the mean knockdown % for each group (PBS control group is considered 100% gene expression, 0% knockdown).

[0447] Tolerability assessment Generally, the tolerability of HSC-LNP was evaluated by intravenous injection of LNP preparations at approximately 0.03–3 mg / kg into female Balb / C mice (5–8 weeks old). Immediately before injection, the siRNA LNP stock solution was filtered and diluted to the required dosage concentration. Two hours after treatment, blood was collected by making a cut in the tail and processed to obtain plasma (for cytokine analysis by ELISA). At the final time point (24 hours after administration), blood (target ≥ 800 μL) was collected by cardiac puncture and sent to IDEXX for CRES 15 panel analysis (CBC / Diff & Clin Chem analysis).

[0448] In short, approximately 300 μL of blood was collected in an EDTA microtainer tube, inverted 10 times to mix with the K2 EDTA whole blood sample, and immediately stored at 4°C to await same-day shipment for hematological analysis. The remaining blood volume (approximately 500 μL) was collected in a serum separation tube, inverted 5 times to mix with the SST sample, and then allowed to coagulate at room temperature for 1 to 1.5 hours. The blood samples were centrifuged to recover the serum for clinical chemical analysis.

[0449] The following siRNA sequence was used from 5' to 3': RELN: S:GGucucAAGccAcucGuuudTsdT AS:AAACGAGUGGCUUGAGACCdTsdT HSP47: S:GAgACACAUGGgUGCuAUUGU AS:AAUAGCACCCAUgUgUCuCAG Legend: Uppercase: Unmodified nucleotide Lowercase: 2'-OMe modifier s:PS (phosphorothioate linkage)

[0450] The following compounds were used in the experiment. Compound(100) [ka] Compound(101) [ka]

[0451] In the experiments described herein and in the figures, the following compound (12) was used as a representative compound of the present invention. [ka]

[0452] As shown in Figure 1, LNPs using the representative compound of the present invention have equivalent potency to the standard despite a reduction in dose to one-quarter (0.025 mg / kg instead of 0.1 mg / kg).

[0453] Figure 2 demonstrates that LNPs using the representative compound of the present invention exhibit high specificity for hepatocytes (HSCs), while LNPs using a comparative lipid designed for delivery to hepatocytes show no knockdown in HSCs. This comparative data indicates that the lipid of the present invention possesses the desired attributes to enable LNP delivery of therapeutic agents to HSCs. LNPs using the representative compound of the present invention demonstrate that maximum knockdown in HSCs can be achieved at approximately 0.03 mg / kg (Figure 3). Furthermore, LNPs using the comparative lipid designed for delivery to hepatocytes require a higher dose (approximately 1 mg / kg) to achieve the same knockdown as the representative lipid of the present invention (0.03 mg / kg) (Figure 4).

[0454] Furthermore, LNP formulated using the representative lipid of the present invention demonstrated tolerability, as it was found that there was no significant increase in liver enzyme levels over a dose range with an upper limit of 3 mg / kg in this study (Figures 5A and 5B).

[0455] LNPs formulated using the representative lipids of the present invention and minimally modified siRNA exhibit a good duration of action (Figure 6).

[0456] Furthermore, LNPs formulated using the representative lipids of the present invention exhibit biodegradability, as they are rapidly removed from mouse liver (Figure 7).

[0457] Table 1 shows further data on various lipids according to the present invention.

[0458] In summary, as demonstrated by the results presented herein, the lipids described herein possess several attributes important for delivering therapeutic agents to hepatic stellate cells, which can be used, for example, for the treatment of hepatic fibrosis, including the ability to deliver therapeutic agents to HSCs, good tolerability, and functional biodegradability. Finally, preferred embodiments of the present invention are described in separate sections. [Embodiment 1] Compound of formula (I): [ka] (In the formula, R 1 C 2 ~C 30 It is hydrocarbyl; R 2 C 2 ~C 30 It is hydrocarbyl; R 3 C 1 ~C 4 Alkyl, or -OC 2 ~C 30 It is hydrocarbyl; R 4 C 2 ~C 30 It is hydrocarbyl; R 5 C 2 ~C 30 It is hydrocarbyl; R6 C 1 ~C 4 Alkyl, or -OC 2 ~C 30 It is hydrocarbyl; X is a divalent linking group. [Embodiment 2] R 1 C 2 ~C 20 The compound according to Embodiment 1, which is a hydrocarbyl. [Embodiment 3] R 1 C 2 ~C 15 The compound according to Embodiment 1, which is a hydrocarbyl. [Embodiment 4] R 1 C 2 ~C 10 The compound according to Embodiment 1, which is a hydrocarbyl. [Embodiment 5] R 1 C 5 ~C 20 The compound according to Embodiment 1, which is a hydrocarbyl. [Embodiment 6] R 1 However, (C 2 ~C 20 ) alkyl, (C 2 ~C 20 ) Alkenil, or (C 2 ~C 20 The compound according to Embodiment 1, which is an alkynyl compound. [Embodiment 7] R 1 (C 7 ~C 15 The compound according to Embodiment 1, which is alkyl. [Embodiment 8] R 1 (C 7 ~C15 The compound according to Embodiment 1, which is an alkenyl. [Embodiment 9] R 1 (C 7 ~C 15 The compound according to Embodiment 1, which is an alkynyl compound. [Embodiment 10] R 1 However, it has only one double bond (C 7 ~C 15 The compound according to Embodiment 1, which is an alkenyl. [Embodiment 11] R 1 The compound according to Embodiment 1, wherein the compound is (Z)-4-decen-1-yl, 1-tridecyl, (Z)-3-hepta-1-yl, or (Z)-5-dodecen-1-yl. [Embodiment 12] R 2 C 2 ~C 20 A compound according to any one of embodiments 1 to 11, which is a hydrocarbyl. [Embodiment 13] R 2 C 2 ~C 15 A compound according to any one of embodiments 1 to 11, which is a hydrocarbyl. [Embodiment 14] R 2 C 2 ~C 10 A compound according to any one of embodiments 1 to 11, which is a hydrocarbyl. [Embodiment 15] R 2 C 5 ~C 20 A compound according to any one of embodiments 1 to 11, which is a hydrocarbyl. [Embodiment 16] R 2 However, (C 2 ~C 20 ) alkyl, (C 2 ~C 20 ) Alkenil, or (C 2 ~C 20 A compound according to any one of embodiments 1 to 11, which is an alkynyl compound. [Embodiment 17] R 2 (C 7 ~C 15 A compound according to any one of embodiments 1 to 11, which is alkyl. [Embodiment 18] R 2 (C 7 ~C 15 A compound according to any one of embodiments 1 to 11, which is an alkenyl. [Embodiment 19] R 2 (C 7 ~C 15 A compound according to any one of embodiments 1 to 11, which is an alkynyl compound. [Embodiment 20] R 2 However, it has only one double bond (C 7 ~C 15 A compound according to any one of embodiments 1 to 11, which is an alkenyl. [Embodiment 21] R 2 The compound according to any of Embodiments 1 to 11, wherein the compound is (Z)-4-decen-1-yl, 1-tridecyl, (Z)-3-hepta-1-yl, or (Z)-5-dodecen-1-yl. [Embodiment 22] R 3 C 1 ~C 4 A compound according to any one of embodiments 1 to 21, which is alkyl. [Embodiment 23] R 3 A compound according to any one of embodiments 1 to 21, wherein the compound is methyl. [Embodiment 24] R 3 ga-OC 2 ~C 20 A compound according to any one of Embodiments 1 to 21, which is a hydrocarbyl. [Embodiment 25] R 3 ga-OC 2 ~C 15 A compound according to any one of Embodiments 1 to 21, which is a hydrocarbyl. [Embodiment 26] R 3 ga-OC 2 ~C 10 A compound according to any one of Embodiments 1 to 21, which is a hydrocarbyl. [Embodiment 27] R 3 ga-OC 5 ~C 20 A compound according to any one of Embodiments 1 to 21, which is a hydrocarbyl. [Embodiment 28] R 3 However, -O-(C 2 ~C 20 )alkyl, -O-(C 2 ~C 20 ) alkenyl, or -O-(C 2 ~C 20 A compound according to any one of embodiments 1 to 21, which is an alkynyl compound. [Embodiment 29] R 3 ga-O-(C 7 ~C 15 A compound according to any one of embodiments 1 to 21, which is alkyl. [Embodiment 30] R 3 ga-O-(C 7 ~C 15 A compound according to any one of embodiments 1 to 21, which is an alkenyl. [Embodiment 31] R 3 ga-O-(C 7 ~C 15 A compound according to any one of embodiments 1 to 21, which is an alkynyl compound. [Embodiment 32] R 3 However, -O-(C) has only one double bond. 7 ~C 15 A compound according to any one of embodiments 1 to 21, which is an alkenyl. [Embodiment 33] R 3 The compound according to any of Embodiments 1 to 21, wherein the compound is (Z)-4-decene-1-yloxy, 1-tridecyloxy, (Z)-3-hepta-1-yloxy, or (Z)-5-dodecene-1-yloxy. [Embodiment 34] R 4 C 2 ~C 20 A compound according to any one of Embodiments 1 to 33, which is a hydrocarbyl. [Embodiment 35] R 4 C 2 ~C 15 A compound according to any one of Embodiments 1 to 33, which is a hydrocarbyl. [Embodiment 36] R 4 C 2 ~C 10 A compound according to any one of Embodiments 1 to 33, which is a hydrocarbyl. [Embodiment 37] R 4 C 5 ~C 20 A compound according to any one of Embodiments 1 to 33, which is a hydrocarbyl. [Embodiment 38] R 4 However, (C 2 ~C 20 ) alkyl, (C 2 ~C 20 ) Alkenil, or (C 2 ~C 20 A compound according to any one of embodiments 1 to 33, which is an alkynyl compound. [Embodiment 39] R 4 (C 7 ~C 15 A compound according to any one of embodiments 1 to 33, which is alkyl. [Embodiment 40] R 4 (C 7 ~C 15 A compound according to any one of embodiments 1 to 33, which is an alkenyl. [Embodiment 41] R 4 (C 7 ~C 15 A compound according to any one of embodiments 1 to 33, which is an alkynyl compound. [Embodiment 42] R 4 However, it has only one double bond (C 7 ~C 15 A compound according to any one of embodiments 1 to 33, which is an alkenyl. [Embodiment 43] R 4 The compound according to any of Embodiments 1 to 33, wherein the compound is (Z)-4-decen-1-yl, 1-tridecyl, (Z)-3-hepta-1-yl, or (Z)-5-dodecen-1-yl. [Embodiment 44] R 5 C 2 ~C 20 A compound according to any one of Embodiments 1 to 43, which is a hydrocarbyl. [Embodiment 45] R 5 C 2 ~C 15 A compound according to any one of Embodiments 1 to 43, which is a hydrocarbyl. [Embodiment 46] R 5 C 2 ~C 10 A compound according to any one of Embodiments 1 to 43, which is a hydrocarbyl. [Embodiment 47] R 5 C 5 ~C 20 A compound according to any one of Embodiments 1 to 43, which is a hydrocarbyl. [Embodiment 48] R 5 However, (C 2 ~C 20 ) alkyl, (C 2 ~C 20 ) Alkenil, or (C 2 ~C 20 A compound according to any one of embodiments 1 to 43, which is an alkynyl compound. [Embodiment 49] R 5 (C 7 ~C 15 A compound according to any one of embodiments 1 to 43, which is alkyl. [Embodiment 50] R 5 (C 7 ~C 15 A compound according to any one of embodiments 1 to 43, which is an alkenyl. [Embodiment 51] R 5 (C 7 ~C 15 A compound according to any one of embodiments 1 to 43, which is an alkynyl compound. [Embodiment 52] R 5 However, it has only one double bond (C 7 ~C 15 A compound according to any one of embodiments 1 to 43, which is an alkenyl. [Embodiment 53] R 5 The compound according to any of Embodiments 1 to 43, wherein the compound is (Z)-4-decen-1-yl, 1-tridecyl, (Z)-3-hepta-1-yl, or (Z)-5-dodecen-1-yl. [Embodiment 54] R 6 C 1 ~C 4 A compound according to any one of embodiments 1 to 53, which is alkyl. [Embodiment 55] R 6 A compound according to any one of embodiments 1 to 53, wherein the compound is methyl. [Embodiment 56] R 6 ga-OC 2 ~C 20 A compound according to any one of Embodiments 1 to 53, which is a hydrocarbyl. [Embodiment 57] R 6 ga-OC 2 ~C 15 A compound according to any one of Embodiments 1 to 53, which is a hydrocarbyl. [Embodiment 58] R 6 ga-OC 2 ~C 10 A compound according to any one of Embodiments 1 to 53, which is a hydrocarbyl. [Embodiment 59] R 6 ga-OC 5 ~C 20 A compound according to any one of Embodiments 1 to 53, which is a hydrocarbyl. [Embodiment 60] R 6 However, -O-(C 2 ~C 20 )alkyl, -O-(C 2 ~C 20 ) alkenyl, or -O-(C 2 ~C 20 A compound according to any one of embodiments 1 to 53, which is an alkynyl compound. [Embodiment 61] R 6 ga-O-(C 7 ~C 15 A compound according to any one of embodiments 1 to 53, which is alkyl. [Embodiment 62] R 6 ga-O-(C 7 ~C 15 A compound according to any one of embodiments 1 to 53, which is an alkenyl. [Embodiment 63] R 6 ga-O-(C 7 ~C 15 A compound according to any one of embodiments 1 to 53, which is an alkynyl compound. [Embodiment 64] R 6 However, -O-(C) has only one double bond. 7 ~C 15 A compound according to any one of embodiments 1 to 53, which is an alkenyl. [Embodiment 65] R 6 The compound is one of the embodiments 1 to 53, wherein the compound is (Z)-4-decene-1-yloxy, 1-tridecyloxy, (Z)-3-hepta-1-yloxy, or (Z)-5-dodecene-1-yloxy. [Embodiment 66] X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, wherein one or more of the carbon atoms (e.g., 1, 2, 3, or 4) are optionally (-O-) or (-NR a -), (divalent (C 3 ~C 8 It is replaced by a cycloalkyl group or a divalent 3-12 membered heterocycle, and the chain, the divalent (C 3 ~C 8 )Cycloalkyl, or the aforementioned divalent 3-12 membered heterocycle, (C 1 ~C 6 )alkoxy, (C 1 ~C6 ) Alkanoyl, (C 1 ~C 6 ) Alkanoyl oxy, (C 1 ~C 6 ) are optionally substituted with one or more substituents (e.g., one, two, three, or four) independently selected from the group consisting of alkoxycarbonyl, cyano, nitro, halo, hydroxy, oxo (=O), and carboxyl, and each R a They became independent, H, and Halo, hydroxy and (C 1 ~C 6 )Optionally substituted with one or more groups independently selected from the alkoxy (C 1 ~C 6 ) alkyl A compound selected from any of embodiments 1 to 65. [Embodiment 67] X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, where one or more of the carbon atoms (e.g., 1, 2, 3, or 4) are optionally (-O-) or (-NR a -), (divalent (C 3 ~C 8 It is replaced by a cycloalkyl group or a divalent 3-8 membered heterocycle, and the chain, the divalent (C 3 ~C 8 )Cycloalkyl, or the aforementioned divalent 3-8 membered heterocycle, (C 1 ~C 6 )alkoxy, (C 1 ~C 6 ) Alkanoyl, (C 1 ~C 6 ) Alkanoyl oxy, (C 1 ~C 6 ) are optionally substituted with one or more substituents (e.g., one, two, three, or four) independently selected from the group consisting of alkoxycarbonyl, cyano, nitro, halo, hydroxy, oxo (=O), and carboxyl, and each R a H and (C 1 ~C 6 A compound selected from alkyl groups, according to any one of embodiments 1 to 65. [Embodiment 68] X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, wherein one or more of the carbon atoms (e.g., 1, 2, 3, or 4) are optionally (-NR a -) or (a divalent 3-12 member heterocycle), and each R a They became independent, Halo, hydroxy and (C 1 ~C 6 )Optionally substituted with one or more groups independently selected from the alkoxy (C 1 ~C 6 ) alkyl A compound selected from any of embodiments 1 to 65. [Embodiment 69] X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, wherein one or more of the carbon atoms (e.g., 1, 2, 3, or 4) are optionally (-NR a -) or (a divalent 3- to 8-membered heterocycle), and each R a (C 1 ~C 6 A compound selected from alkyl groups, according to any one of embodiments 1 to 65. [Embodiment 70] X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, wherein one or more of the carbon atoms (e.g., 1, 2, 3, or 4) are (-NR a -) is replaced, and each R a They became independent, Halo, hydroxy and (C 1 ~C 6 )Optionally substituted with one or more groups independently selected from the alkoxy (C 1 ~C 6 ) alkyl A compound selected from any of embodiments 1 to 65. [Embodiment 71] X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, wherein one or more of the carbon atoms (e.g., 1, 2, 3, or 4) are (-NR a -) is replaced, and each R a (C 1 ~C 6 A compound selected from alkyl groups, according to any one of embodiments 1 to 65. [Embodiment 72] The compound according to any one of Embodiments 1 to 65, wherein X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, and one or more of the carbon atoms (e.g., one, two, three, or four) are optionally replaced by a divalent 3 to 12-membered heterocycle. [Embodiment 73] The compound according to any one of Embodiments 1 to 65, wherein X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, and one or more of the carbon atoms (e.g., one, two, three, or four) are optionally replaced by a divalent 3 to 8-membered heterocycle. [Embodiment 74] The compound according to any one of Embodiments 1 to 65, wherein X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 6 to 12 carbon atoms, and one or more of the carbon atoms (e.g., one, two, three, or four) are optionally replaced by piperazine-1,4-diyl. [Embodiment 75] X

change

change

change

change

change

change

change

change

change

Claims

1. Compound of formula (I): 【Chemistry 1】 [In the formula, R １ is a linear C ７ ~C ３０ It is hydrocarbyl; R ２ is a linear C ７ ~C ３０ It is hydrocarbyl; R ３ is C １ to C ４ alkyl, or -O-C ２ to C ３０ hydrocarbyl; R ４ is a linear C ７ ~C ３０ It is hydrocarbyl; R ５ is a linear C ７ ~C ３０ It is hydrocarbyl; R ６ C １ ~C ４ Alkyl, or -O-C ２ ~C ３０ It is hydrocarbyl; X is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms. One or more carbon atoms in the hydrocarbon chain are independently -NR ａ - or replaced by a divalent 3-12 membered heterocycle having 1-3 nitrogen atoms, One or more carbon atoms in the hydrocarbon chain are optionally -O- or divalent (C ３ ~C ８ ) It has been replaced by a cycloalkyl group, The hydrocarbon chain, the divalent (C ３ ~C ８ ) Cycloalkyl, or the aforementioned divalent 3-12 membered heterocycle, optionally, (C １ ~C ６ ) Alkoxy, (C １ ~C ６ ) Alkanoyl, (C １ ~C ６ ) Alkanoyl oxy, (C １ ~C ６ ) Substituted with one or more substituents independently selected from the group consisting of alkoxycarbonyl, cyano, nitro, halo, hydroxy, oxo, and carboxyl, Each R ａ H and (C) are independent of each other. １ ~C ６ [Selected from alkyl groups].

2. R １ However, linear C ７ ~C ２０ hydrocarbil, and / or R ２ However, linear C ７ ~C ２０ hydrocarbil, and / or R ３ However, C １ ~C ４ Alkyl, or -O-C ２ ~C ２０ hydrocarbil, and / or R ４ However, linear C ７ ~C ２０ hydrocarbil, and / or R ５ However, linear C ７ ~C ２０ hydrocarbil, and / or R ６ However, C １ ~C ４ Alkyl, or -O-C ２ ~C ２０ It is hydrocarbil. The compound according to claim 1.

3. R １ However, linear (C ７ ~C ２０ ) alkyl, linear (C ７ ~C ２０ ) Alkenyl, or linear (C ７ ~C ２０ ) is alkinyl, and / or R ２ However, linear (C ７ ~C ２０ ) alkyl, linear (C ７ ~C ２０ ) Alkenyl, or linear (C ７ ~C ２０ ) is alkinyl, and / or R ３ However, C １ ~C ４ Alkyl, O-(C ２ ~C ２０ ) alkyl, -O-(C ２ ~C ２０ ) alkenyl, or -O-(C ２ ~C ２０ ) is alkinyl, and / or R ４ is a linear (C ７ -C ２０ ) alkyl, linear (C ７ -C ２０ ) alkenyl, or linear (C ７ -C ２０ ) alkynyl, and / or R ５ is a linear (C ７ ~C ２０ ) alkyl, linear (C ７ ~C ２０ ) alkenyl, or linear (C ７ ~C ２０ ) alkynyl, and / or R ６ However, C １ ~C ４ Alkyl, -O-(C ２ ~C ２０ ) alkyl, -O-(C ２ ~C ２０ ) alkenyl, or -O-(C ２ ~C ２０ ) is alkinyl, The compound according to claim 1.

4. R １ However, a straight chain (C) has only one double bond. ７ ~C １５ ) are alkenils, and / or R ２ However, a straight chain (C) has only one double bond. ７ ~C １５ ) are alkenils, and / or R ３ However, it has only one methyl or double bond -O-(C ７ ~C １５ ) are alkenils, and / or R ４ However, a straight chain (C) has only one double bond. ７ ~C １５ ) are alkenils, and / or R ５ However, a straight chain (C) has only one double bond. ７ ~C １５ ) are alkenils, and / or R ６ However, it has only one methyl or double bond -O-(C ７ ~C １５ ) is Alkenil, The compound according to claim 1.

5. X, 【Chemistry 2】 (In the formula, a is 2, 3, 4, or 5; b is 2, 3, 4, or 5; c is 2, 3, 4, or 5; d is 2, 3, 4, or 5; e is 2, 3, 4, 5, 6, 7, or 8; f is 2, 3, 4, 5, 6, 7, or 8; Each R ａ H and (C １ ~C ６ (Selected from alkyl) A compound according to any one of claims 1 to 4, selected from the group consisting of the following.

6. X, 【Transformation 3】 A compound according to any one of claims 1 to 4, selected from the group consisting of the following.

7. A compound according to claim 1, or a salt thereof, selected from compounds 12-32, 34-38, and 40-43 shown in the table below. Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 Table 1-7

8. Lipid particles comprising the compound described in any one of claims 1 to 7.

9. The lipid particles according to claim 8, further comprising a therapeutic agent.

10. Lipid particles according to claim 9, wherein the therapeutic agent is a nucleic acid therapeutic agent.

11. The lipid particles according to claim 10, wherein the nucleic acid therapeutic agent is an interfering RNA agent or mRNA.

12. The lipid particle according to claim 11, wherein the nucleic acid therapeutic agent is siRNA.

13. A composition comprising the compound described in any one of claims 1 to 7.

14. A pharmaceutical composition comprising lipid particles according to any one of claims 8 to 12, and a pharmaceutically acceptable carrier.

15. Lipid particles according to any one of claims 9 to 12 for use in vivo delivery of therapeutic agents to mammals.

16. Use of lipid particles according to any one of claims 9 to 12 for preparing a pharmaceutical for in vivo delivery of a therapeutic agent to a mammal.

17. A composition for treating a disease or disorder in a mammalian subject in need, comprising lipid particles according to any one of claims 9 to 12.

18. The composition according to claim 17, wherein the disease or disorder is hepatic fibrosis, non-alcoholic steatohepatitis (NASH), or alcoholic steatohepatitis (ASH).

19. The composition according to claim 17, wherein the disease or disorder is non-alcoholic steatohepatitis (NASH) or alcoholic steatohepatitis (ASH) associated with hepatic fibrosis.

20. A composition for delivering a therapeutic agent to hepatic stellate cells (HSCs) in vivo or in vitro, comprising lipid particles according to any one of claims 9 to 12, wherein the lipid particles are brought into contact with the HSCs.

Images