Cationic Lipids for Lipid Nanoparticle Delivery of Therapeutic Agents to Hepatic Stellate Cells

Abstract

Certain embodiments of the present invention provide lipids useful for preparing lipid nanoparticles for delivery of therapeutic agents (eg, to hepatic stellate cells).

Description

Cross - reference to related applications

[0001] This application claims the benefit of priority to U.S. Patent Application No. 62 / 990,939, filed on March 17, 2020, which is incorporated herein by reference.

Background Art

[0002] Nanoparticles containing cationic lipids have been used for the delivery of various therapeutic agents. Currently, due to the difficulties and costs associated with the preparation of the cationic lipid components of these nanoparticles, their attractiveness for commercial development as delivery vehicles may be limited. Accordingly, there is a need for additional cationic lipids that can be incorporated into lipid nanoparticles. For example, there is a need for cationic lipids that can be prepared using a more inexpensive and efficient process. There is also a need for lipid nanoparticles having characteristics for delivering therapeutic agents (such as nucleic acids) to hepatic stellate cells for treatment (e.g., treatment of liver fibrosis).

Summary of the Invention

Means for Solving the Problems

[0003] The present invention provides a cationic lipid that is effective in delivering an active agent or therapeutic agent (such as a nucleic acid) to hepatic stellate cells when incorporated into lipid nanoparticles.

[0004] Accordingly, in certain embodiments, Formula (I):

Chemical Formula

[0005] Also provided are lipid particles comprising such compounds, and methods of using the lipid particles (e.g., for specifically delivering a therapeutic agent to hepatic stellate cells to treat a disease).

Brief Description of the Drawings

[0006]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0007] In certain embodiments, formula (I):

Chemical formula

[0008] In certain embodiments, each R a and R b is independently selected from the group consisting of (C1-C6) alkyl substituted by one or more groups independently selected from halo and hydroxy and H.

[0009] In certain embodiments, each R a and R b is independently selected from the group consisting of H and (C1-C6) alkyl, and at least one of R a and R b is substituted by halo.

[0010] In certain embodiments, each R a and R b is independently selected from the group consisting of H and (C1-C6) alkyl, and at least one of R a and R b is substituted by hydroxy.

[0011] In certain embodiments, R 1 is (C5-C 25 ) alkyl.

[0012] In certain embodiments, R 1 is (C5-C 25 ) alkenyl.

[0013] In certain embodiments, R 1 is (C5-C 25 ) alkynyl.

[0014] In certain embodiments, R 1 is (C5-C 20 ) alkyl.

[0015] In certain embodiments, R 1 is (C5-C 20 ) alkenyl.

[0016] In certain embodiments, R 1 is (C5-C 20 ) alkynyl.

[0017] In certain embodiments, R 1 is (C 10 -C 20 ) alkyl.

[0018] In certain embodiments, R 1 is (C 10 -C 20 ) alkenyl.

[0019] In certain embodiments, R 1 is (C 10 -C 20 ) alkynyl.

[0020] In certain embodiments, R 1 is 4-decen-1-yl or 8,10-heptadecadien-1-yl.

[0021] In certain embodiments, R 2 is (C5-C 25 ) alkyl.

[0022] In certain embodiments, R 2 is (C5-C 25 ) alkenyl.

[0023] In certain embodiments, R 2 is (C5-C 25 ) alkynyl.

[0024] In certain embodiments, R 2 is (C5-C 20 ) alkyl.

[0025] In certain embodiments, R 2 is (C5-C 20 ) alkenyl.

[0026] In certain embodiments, R 2 is (C5-C 20 ) alkynyl.

[0027] In certain embodiments, R 2 is (C 10 -C 20 ) alkyl.

[0028] In certain embodiments, R 2 is (C 10 -C 20 ) alkenyl.

[0029] In certain embodiments, R 2 is (C 10 -C 20 ) alkynyl.

[0030] In certain embodiments, R 2 is 4-decen-1-yl.

[0031] In certain embodiments, R 3 is (C5-C 25 ) alkyl.

[0032] In certain embodiments, R 3 is (C5-C 25 ) alkenyl.

[0033] In certain embodiments, R 3 is (C5-C25 ) is alkynyl.

[0034] In certain embodiments, R 3 is (C5-C 20 ) alkyl.

[0035] In certain embodiments, R 3 is (C5-C 20 ) alkenyl.

[0036] In certain embodiments, R 3 is (C5-C 20 ) alkynyl.

[0037] In certain embodiments, R 3 is (C 10 -C 20 ) alkyl.

[0038] In certain embodiments, R 3 is (C 10 -C 20 ) alkenyl.

[0039] In certain embodiments, R 3 is (C 10 -C 20 ) alkynyl.

[0040] In certain embodiments, R 3 is 4-decen-1-yl.

[0041] In certain embodiments, R 4 is (C3-C a R b ) alkyl substituted by one or more groups independently selected from chloro, bromo, iodo, and -NR 15 ).

[0042] In certain embodiments, R 4 is (C3-C a R b ) alkyl substituted by one or more groups independently selected from chloro, bromo, iodo, and -NR15 ) is alkenyl.

[0043] In certain embodiments, R 4 is (C3-C a R b ) alkynyl substituted by one or more groups independently selected from chloro, bromo, iodo, and -NR 15 ).

[0044] In certain embodiments, R 4 is (C3-C a R b ) alkyl substituted by one or more groups independently selected from chloro, bromo, iodo, and -NR 10 ).

[0045] In certain embodiments, R 4 is (C3-C a R b ) alkenyl substituted by one or more groups independently selected from chloro, bromo, iodo, and -NR 10 ).

[0046] In certain embodiments, R 4 is (C3-C a R b ) alkynyl substituted by one or more groups independently selected from chloro, bromo, iodo, and -NR 10 ).

[0047] In certain embodiments, R 4 is (C3-C 10 ) alkyl, (C3-C 10 ) alkenyl, or (C3-C 10 ) alkynyl, and the (C3-C 10 ) alkyl, (C3-C 10 ) alkenyl, and C3-C 10 ) alkynyl are substituted by one or more groups independently selected from chloro, bromo, iodo, and -NR a R b ).

[0048] In certain embodiments, R 4 is (C3-C 15 ) alkyl, (C3-C 15 ) alkenyl, or (C3-C 15 ) alkynyl, and the (C3-C 15 ) alkyl, (C3-C 15 ) alkenyl, and C3-C 15 ) is substituted by chloro, bromo, or iodo.

[0049] In certain embodiments, R 4 is (C3-C 15 ) alkyl, (C3-C 15 ) alkenyl, or (C3-C 15 ) alkynyl, and the (C3-C 15 ) alkyl, (C3-C 15 ) alkenyl, and C3-C 15 ) alkynyl is substituted by -NR a R b .

[0050] In certain embodiments, each R a and R b is independently selected from the group consisting of (C1-C6) alkyl.

[0051] In certain embodiments, each R a and R b is substituted by one or more groups independently selected from halo and hydroxy.

[0052] In certain embodiments, at least one of R a and R b is substituted by halo.

[0053] In certain embodiments, at least one of R a and R b is substituted by hydroxy.

[0054] In certain embodiments, each R a and R bis methyl.

[0055] In certain embodiments, R 4 is 5-(N,N-dimethylamino)penta-1-yl.

[0056] In certain embodiments, the compound: [Chemical formula] or a salt thereof is provided herein, wherein each R a and R b is independently selected from the group consisting of (C1-C6)alkyl optionally substituted by one or more groups independently selected from halo and hydroxy and H, or R a and R b together with the nitrogen to which they are attached form a ring selected from the group consisting of aziridine, azetidine, pyrrolidine, piperidine, piperazine, morpholino, and thiomorpholino, and the ring is optionally substituted by one or more groups independently selected from (C1-C6)alkyl.

[0057] In certain embodiments, each R a and R b is independently selected from the group consisting of (C1-C6)alkyl substituted by one or more groups independently selected from halo and hydroxy and H.

[0058] In certain embodiments, at least one of R a and R b is substituted by halo.

[0059] In certain embodiments, at least one of R a and R b is substituted by hydroxy.

[0060] In certain embodiments, a compound selected from the compounds described herein or a salt thereof is provided herein.

[0061] In certain embodiments, lipid particles comprising the compounds described herein are provided herein.

[0062] In certain embodiments, the compound is selected from the compounds described in the examples.

[0063] Also provided herein are lipid particles comprising the compounds described herein.

[0064] In certain embodiments, the lipid particles further comprise a non-cationic lipid.

[0065] In certain embodiments, the lipid particles further comprise a conjugate lipid that suppresses aggregation of the particles.

[0066] In certain embodiments, the lipid particles further comprise a therapeutic agent.

[0067] In certain embodiments, the therapeutic agent is a nucleic acid therapeutic agent.

[0068] In certain embodiments, the therapeutic agent is an interfering RNA agent.

[0069] In certain embodiments, the therapeutic agent is siRNA.

[0070] In certain embodiments, the therapeutic agent is mRNA.

[0071] In certain embodiments, the nucleic acid therapeutic agent comprises at least one modified nucleotide.

[0072] In certain embodiments, the nucleic acid comprises at least one 2'-O-methyl (2'OMe) nucleotide.

[0073] In certain embodiments, the non-cationic lipid is cholesterol or a derivative thereof.

[0074] In certain embodiments, the non-cationic lipid is cholesterol.

[0075] In certain embodiments, the non-cationic lipid comprises a phospholipid.

[0076] In certain embodiments, the non-cationic lipid comprises a mixture of a phospholipid and cholesterol.

[0077] In certain embodiments, the phospholipid is distearoyl phosphatidylcholine (DSPC).

[0078] In certain embodiments, the conjugate lipid is a polyethylene glycol (PEG)-lipid conjugate.

[0079] In certain embodiments, the PEG-lipid conjugate is a PEG-dimyristyloxypropyl (PEG-DMA) conjugate.

[0080] Also provided herein are compositions comprising the compounds or lipid particles described herein.

[0081] Also provided herein are pharmaceutical compositions comprising the compounds or lipid particles described herein and a pharmaceutically acceptable carrier.

[0082] Also provided is a method for in vivo delivery of a therapeutic agent, the method comprising administering the lipid particles described herein to a mammalian subject.

[0083] Also provided are the lipid particles described herein for use in in vivo delivery of a therapeutic agent to a mammal.

[0084] Also provided is the use of the lipid particles described herein for preparing a medicament for in vivo delivery of a therapeutic agent to a mammal.

[0085] Also provided is a method for treating a disease or disorder in a mammalian subject in need of treatment thereof, the method comprising administering to the mammalian subject a therapeutically effective amount of the lipid particles described herein.

[0086] In certain embodiments, the disease or disorder is liver fibrosis.

[0087] In certain embodiments, the disease or disorder is non-alcoholic steatohepatitis (NASH).

[0088] In certain embodiments, the disease or disorder is alcoholic steatohepatitis (ASH).

[0089] In certain embodiments, the disease or disorder is non-alcoholic steatohepatitis (NASH) or alcoholic steatohepatitis (ASH) associated with liver fibrosis.

[0090] Also provided is a method of delivering a therapeutic agent to hepatic stellate cells (HSCs) in vivo or in vitro, the method comprising contacting the HSCs with the lipid particles described herein.

[0091] Liver fibrosis is caused by the excessive accumulation of extracellular matrix during chronic liver injury. Activation of hepatic stellate cells (HSCs) is an important step during liver fibrosis. Targeted delivery of therapeutic agents to HSCs (e.g., activated HSCs) can be important for the successful treatment of liver fibrosis. Several protein markers have been shown to be overexpressed in activated HSCs, and their ligands have been used to specifically deliver various antifibrotic agents (see, for example, Chen et al., Journal of Pharmacology and Experimental Therapeutics, 2019, 370(3)695-702). However, the use of other systems (such as lipid nanoparticles (LNPs)) to deliver therapeutic agents is needed as another means of delivering therapeutic agents to HSCs.

[0092] Liver fibrosis is caused by the abnormal formation of a large amount of scar tissue in the liver. Liver fibrosis occurs when the liver attempts to repair and replace damaged cells. Various disorders and drugs can damage the liver and cause fibrosis.

[0093] Non-alcoholic fatty liver disease (NAFLD) is a condition in which triglycerides accumulate in the liver. Non-alcoholic steatohepatitis (NASH) is a type of NAFLD. NASH is associated with inflammatory changes and hepatocyte injury. NASH is a major cause of liver disease and often progresses to liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) have similar pathogenesis and histopathology but different etiologies and epidemiology. NASH and ASH are advanced stages of non-alcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (AFLD). Alcoholic steatohepatitis (ASH) is a chronic and progressive liver disease characterized by liver fibrosis and potentially liver tissue necrosis, which is caused by excessive and long-term alcohol consumption. Women are more prone to this disease because they have lower alcohol metabolism compared to men.

[0094] Liver fibrosis is an important underlying cause of liver dysfunction and is predictive of mortality. As it progresses to cirrhosis and HCC and ultimately leads to liver failure, liver transplantation becomes necessary. The current number of patients with NASH-related fibrosis (F2 and above) in the United States is approximately 3.8 million. In treating NAFLD and NASH, physicians have typically recommended weight loss. While weight loss can reduce fat, inflammation, and fibrosis in the liver, there are no drugs approved for the treatment of NAFLD and NASH. Specifically, there are no drugs approved for the treatment of liver 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 therapeutic treatment options, including delivery options, are needed for the treatment of liver fibrosis (such as those associated with NASH or ASH).

[0095] Definitions Unless otherwise indicated, the following definitions are used: Alkyl, alkenyl, alkynyl, etc. refer to both straight-chain and branched-chain groups, but references to individual radicals (such as propyl) include only straight-chain radicals, and branched-chain isomers (such as isopropyl) are specifically mentioned.

[0096] The term "alkyl", alone or as part of another substituent, means a straight-chain or branched-chain hydrocarbon radical having the specified number of carbon atoms (i.e., C 5-25 means 5 to 25 carbons). Examples include (C5-C 20 ) alkyl, (C3-C 15 ) alkyl, (C 10 -C 20 ) alkyl, and (C3-C 10) Alkyl is exemplified. In one embodiment, the alkyl group has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, iso-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and higher homologs and isomers.

[0097] The term "alkenyl" refers to an unsaturated alkyl radical having one or more (e.g., 1, 2, 3, or 4) double bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), and higher homologs and isomers. In one embodiment, the alkene is 4-decen-1-yl. In one embodiment, the alkene is 4-decen-1-yl or 8,10-heptadecadien-1-yl.

[0098] The term "alkynyl" refers to an unsaturated alkyl radical having one or more (e.g., 1, 2, 3, or 4) triple bonds and optionally one or more double bonds. In one embodiment, the alkynyl has one or more triple bonds and no double bonds. In another embodiment, the alkynyl has one or more (e.g., 1, 2, 3, or 4) triple bonds and one or more (e.g., 1, 2, 3, or 4) double bonds. Examples of such unsaturated alkyl groups include ethynyl, 1-propynyl and 3-propynyl, 3-butynyl, and higher homologs and isomers.

[0099] The term "alkoxy" refers to an alkyl group bonded to the remainder of the molecule through an oxygen atom ("oxy").

[0100] The term "cycloalkyl" refers to a saturated or partially unsaturated (non-aromatic) ring having 3 to 8 carbon atoms, all of which are carbon (e.g., a (C3-C8) carbocyclic ring).

[0101] The term "heterocyclic" refers to a saturated or partially unsaturated monocyclic ring having at least one atom other than carbon in the ring, and the atom is selected from the group consisting of oxygen, nitrogen, and sulfur. Thus, this term includes a saturated or partially unsaturated monocyclic ring (e.g., a 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, or 8-membered ring) having about 1 to 6 carbon atoms and about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur in the ring. Sulfur atoms and nitrogen atoms may also exist in their oxidized forms. Examples of heterocycles include, but are not limited to, azetidinyl, tetrahydrofuranyl, piperazinyl, and piperidinyl.

[0102] As used herein, the term "alkoxycarbonyl" refers to the group (alkyl)-O-C(=O)-, wherein the term alkyl has the meaning defined herein.

[0103] As used herein, the term "alkanoyloxy" refers to the group (alkyl)-C(=O)-O-, wherein the term alkyl has the meaning defined herein.

[0104] As used herein, the term "heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

[0105] As used herein, intersecting a bond in a chemical structure The wavy line TIFF0007709981000004.tif15115 refers to the attachment point of the bond where the wavy bond intersects the remainder of the molecule in the chemical structure.

[0106] 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 can reduce or inhibit the expression of a target gene or sequence (e.g., by mediating the degradation of mRNA complementary to the interfering RNA sequence or by inhibiting its translation) when the interfering RNA is present in the same cell as the target gene or sequence. Thus, interfering RNA refers to single-stranded RNA complementary to the target mRNA sequence, or double-stranded RNA formed by two complementary strands or by a single self-complementary strand. The interfering RNA may have substantial or complete identity to the target gene or sequence, or may contain mismatch regions (i.e., mismatch motifs). The sequence of the interfering RNA may correspond to the full-length target gene or a partial sequence thereof.

[0107] Interfering RNAs include "small interfering RNAs" or "siRNAs", for example, interfering RNAs having a length of about 15 to 60, 15 to 50, or 15 to 40 (double-stranded) nucleotides, more typically about 15 to 30, 15 to 25, or 19 to 25 (double-stranded) nucleotides in length, preferably about 20 to 24, 21 to 22, or 21 to 23 (double-stranded) nucleotides in length (for example, each complementary sequence of the double-stranded siRNA is 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 25, or 19 to 25 nucleotides in length, preferably about 20 to 24, 21 to 22, or 21 to 23 nucleotides in length, and the double-stranded siRNA is about 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 25, or 19 to 25 base pairs in length, preferably about 18 to 22, 19 to 20, or 19 to 21 base pairs in length). The siRNA duplex can include a 3' overhang of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and a 5' phosphate terminus. Examples of siRNAs include double-stranded polynucleotide molecules assembled from two separate strand molecules (where one strand is the sense strand and the other is the complementary antisense strand); double-stranded polynucleotide molecules assembled from a single-strand molecule (where the sense region and the antisense region 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 circular single-stranded polynucleotide molecules having two or more loop structures and a stem with self-complementary sense and antisense regions (where this circular polynucleotide can be processed in vivo or in vitro to produce an active double-stranded siRNA molecule), but are not limited thereto.

[0108] Preferably, the siRNA is chemically synthesized. The siRNA can also be produced by cleaving longer dsRNAs (e.g., dsRNAs longer than about 25 nucleotides) with E. coli RNase III or Dicer. These enzymes process the 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 in length to about 100, 200, 300, 400 or 500 nucleotides in length. The dsRNA can be 1000, 1500, 2000, 5000 nucleotides in length or longer. The dsRNA can encode the entire gene transcript or a portion of the gene transcript. In certain cases, the siRNA can be encoded by a plasmid (e.g., transcribed as a sequence that spontaneously folds into a double-stranded hairpin loop).

[0109] As used herein, the terms "mismatch motif" or "mismatch region" refer to the portion of an interfering RNA (e.g., siRNA, aiRNA, miRNA) sequence that does not have 100% complementarity to its target sequence. The interfering RNA can have at least one, two, three, four, five, six, or more mismatch regions. The mismatch regions can be contiguous or separated by one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more nucleotides. The mismatch motif or mismatch region can comprise one nucleotide or can comprise two, three, four, five, or more nucleotides.

[0110] An "effective amount" or "therapeutically effective amount" of an active or therapeutic agent, such as a nucleic acid (e.g., interfering RNA or mRNA), is an amount sufficient to produce a desired effect, e.g., inhibition of the expression of a target sequence as compared to the normal expression level detected in the absence of the interfering RNA, or to produce mRNA-specific expression of an amount of protein that is expressed in vivo and produces a desired biological effect. Inhibition of the expression of a target gene or target sequence is achieved when the value obtained using the interfering RNA is about 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 that is normally expressed in a cell type in the body, and a therapeutically effective amount of mRNA is an amount that produces at least 50% (e.g., at least 60%, or at least 70%, or at least 80%, or at least 90%) of the amount of the encoded protein that is normally expressed in that cell type in a healthy individual. Assays suitable for measuring the expression of a target gene or target sequence include, for example, tests at the protein or RNA level using techniques known to those of skill in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those of skill in the art.

[0111] "Reducing," "reduction," "lowering," or "decrease" of an immune response by interfering RNA is intended to mean a detectable decrease in the immune response to a given interfering RNA (e.g., modified interfering RNA). The amount of decrease in the immune response by the modified interfering RNA can be measured in comparison to the level of the immune response in the presence of unmodified interfering RNA. The detectable decrease can be about 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 decrease in the immune response to interfering RNA is typically measured by a decrease in cytokine production (e.g., IFNγ, IFNα, TNFα, IL-6 or IL-12) by responder cells in vitro, or by a decrease in cytokine production in the serum of a mammalian subject after administration of interfering RNA.

[0112] "Reducing," "reduction," "lowering," or "decrease" of an immune response by mRNA is intended to mean a detectable decrease in the immune response to a given mRNA (e.g., modified mRNA). The amount of decrease in the immune response by the modified mRNA can be measured in comparison to the level of the immune response in the presence of unmodified mRNA. The detectable decrease can be about 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 decrease in the immune response to mRNA is typically measured by a decrease in cytokine production (e.g., IFNγ, IFNα, TNFα, IL-6 or IL-12) by responder cells in vitro, or by a decrease in cytokine production in the serum of a mammalian subject after administration of mRNA.

[0113] As used herein, the term "responder cell" refers to a cell, preferably a mammalian cell, that gives rise to a detectable immune response when contacted with an immunostimulatory interfering RNA such as unmodified siRNA. Exemplary responder cells include, for example, dendritic cells, macrophages, peripheral blood mononuclear cells (PBMCs), splenocytes, and the like. Detectable immune responses include, for example, the production of cytokines or growth factors such as TNF-α, IFN-α, IFN-β, IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TGF, and combinations thereof.

[0114] "Substantial identity" refers to a sequence that hybridizes to a reference sequence under stringent conditions or has a specified percentage of identity over a particular region of the reference sequence.

[0115] The phrase "stringent hybridization conditions" refers to conditions under which a nucleic acid will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences will hybridize particularly at higher temperatures. A wide variety of guidelines for nucleic acid hybridization are found in Tijssen, Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10 °C lower than the thermal melting point (T m ) of the particular sequence at a defined ionic strength and pH. T 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 in excess, the T m at which 50% of the probe is occupied at equilibrium. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, the positive signal is at least 2-fold, preferably 10-fold, that of background hybridization.

[0116] Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5×SSC, and 1% SDS, incubated at 42 °C, or 5×SSC, 1% SDS, incubated at 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 about 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).

[0117] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This occurs, for example, when a copy of the nucleic acid is generated using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and washing in 1× SSC at 45°C. Positive hybridization is at least 2-fold above background. One of ordinary skill in the art will readily appreciate that alternative hybridization and wash conditions can be utilized to obtain similar stringency conditions. Additional guidelines for determining hybridization parameters are provided in numerous references, such as Current Protocols in Molecular Biology, Ausubel et al., eds.

[0118] The terms "substantially identical" or "substantial identity" in the context of two or more nucleic acids refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms, or by manual alignment and visual inspection, have the same or a specified percentage of nucleotides that are the same (e.g., at least about 60%, preferably at least about 65%, 70%, 75%, 80%, 85%, 90% or 95% identity over a specified region). This definition also refers to the complement of the sequence, when 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.

[0119] For array comparison, usually one array serves as a reference array to be compared with a test array. When using an array comparison algorithm, the test array and the reference array are input into a computer, and if necessary, partial array coordinates are specified to specify the array algorithm program parameters. Initial set program parameters may be used, or alternative parameters may be specified. Then, the array comparison algorithm calculates the percent identity of the test array to the reference array based on the program parameters.

[0120] As used herein, a "comparison window" includes reference to any one segment of a number of contiguous positions selected from the group consisting of about 5 to about 60, usually about 10 to about 45, more usually about 15 to about 30, which can be used to compare an array with a reference array of the same number of contiguous positions after the two arrays are optimally aligned. Methods of array alignment for comparison are well known in the art. Optimal array 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 the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds. (1995 supplement)).

[0121] Preferred examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res., 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol., 215:403-410 (1990), respectively. To determine the percent sequence identity of the nucleic acids of the present invention, BLAST and BLAST 2.0 are used with the parameters described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0122] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., 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 indication of the probability that a match between two nucleotide sequences occurs by chance. For example, if the minimum sum probability in a comparison of a test nucleic acid and a 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 to be similar to the reference sequence.

[0123] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, including DNA and RNA. DNA can be, for example, in the form of 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 of these groups. RNA can be in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, natural, and non-natural and have binding properties similar to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2'-O-methyl ribonucleotide, and peptide-nucleic acid (PNA). Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences in addition to the explicitly recited sequence. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res., 19:5081 (1991), Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985), Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked to each other via phosphate groups."Base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which non-limitingly include modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0124] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that includes a partial-length or full-length coding sequence necessary for the production of a polypeptide or precursor polypeptide.

[0125] As used herein, "gene product" refers to the product of a gene, such as an RNA transcript or polypeptide.

[0126] The term "lipid" non-limitingly refers to a group of organic compounds that include esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. They are usually classified into at least three classes: (1) "simple lipids" that include fats, oils, and waxes; (2) "complex lipids" that include phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0127] 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 that prevent particle aggregation) and nucleic acids, where the nucleic acid (e.g., siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, mRNA, self-amplifying RNA, or plasmid (including plasmids from which interfering RNA or mRNA is transcribed)) is encapsulated within the lipid. In one embodiment, the nucleic acid is at least 50% encapsulated in the lipid, in one embodiment, the nucleic acid is at least 75% encapsulated in the lipid, in one embodiment, the nucleic acid is at least 90% encapsulated in the lipid, and in one embodiment, the nucleic acid is completely encapsulated in the lipid. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid conjugate (e.g., a PEG-lipid conjugate). LNPs can exhibit an extended circulation lifetime after intravenous (i.v.) injection, can accumulate at distal sites (e.g., sites physically separated from the administration site), and can mediate the expression of transfected genes or the silencing of target gene expression at these distal sites, making them extremely useful for systemic applications.

[0128] The lipid particles of the present invention (e.g., LNPs) 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. Further, the nucleic acid, when present in the lipid particles of the present invention, is resistant to nuclease degradation in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Application Publication Nos. 20040142025 and 20070042031, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

[0129] As used herein, "lipid encapsulated" can refer to lipid particles that provide an active or therapeutic agent, such as a nucleic acid (e.g., interfering RNA or mRNA), that is either fully encapsulated, partially encapsulated, or both. In a preferred embodiment, the nucleic acid is fully encapsulated within the lipid particles (e.g., to form SPLP, pSPLP, LNP, or other nucleic acid-lipid particles).

[0130] The term "lipid conjugate" refers to a conjugated lipid that inhibits 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, e.g., U.S. Patent No. 5,885,613, which is hereby incorporated by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. The PEG may be conjugated directly to the lipid or linked to the lipid via a linker moiety. For example, any linker moiety suitable for coupling PEG to a lipid can be used, including ester-free linker moieties and ester-containing linker moieties. In a preferred embodiment, an ester-free linker moiety is used.

[0131] The term "amphiphilic lipid" refers, in part, to any suitable substance in which the hydrophobic portion of the lipid material is oriented in a hydrophobic phase while the hydrophilic portion is oriented in an aqueous phase. The hydrophilic character is due to the presence of polar or charged groups such as carbohydrates, phosphates, carboxyls, sulfatos, aminos, sulfhydryls, nitros, hydroxyls, and other similar groups. Hydrophobicity can be imparted by including nonpolar groups such as long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted by one or more aromatic groups (plural possible), alicyclic groups (plural possible), or heterocyclic groups (plural possible), but not limited thereto. Examples of amphiphilic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0132] 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 compounds lacking phosphorus such as sphingolipids, the sphingoglycolipid family, diacylglycerol, and β-acyl oxy acids are also included in the group designated as amphiphilic lipids. Further, the aforementioned amphiphilic lipids can be mixed with other lipids including triglycerides and sterols.

[0133] The term "neutral lipid" refers to any of several lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol.

[0134] The term "non-cationic lipid" refers to any amphiphilic lipid and any other neutral or anionic lipid.

[0135] 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.

[0136] The term "cationic lipid" refers to the compounds of formula (I) described herein.

[0137] The term "hydrophobic lipid" refers to compounds having nonpolar groups that include long-chain saturated and unsaturated aliphatic hydrocarbon groups and optionally one or more aromatic groups, cycloaliphatic groups, or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0138] The term "membrane fusion" refers to the ability of lipid particles such as LNPs to fuse with the membrane of a cell. The membrane can be either the plasma membrane or the membrane surrounding an organelle, such as an endosome, nucleus, etc.

[0139] As used herein, the term "aqueous solution" refers to a composition that contains water, either wholly or in part.

[0140] As used herein, the term "organic lipid solution" refers to a composition that contains an organic solvent having a lipid, either wholly or in part.

[0141] As used herein, the term "distal site" refers to a physically separated site that includes sites widely distributed throughout the body, not limited to adjacent capillary beds.

[0142] "Serum stability" associated with 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.

[0143] As used herein, "systemic delivery" refers to the delivery of lipid particles that results in widespread in vivo distribution of an active or therapeutic agent such as interfering RNA or mRNA in the body. There are administration techniques that can result in systemic delivery of a particular drug, and those that cannot. Systemic delivery means that a useful amount, preferably a therapeutic amount, of the drug is exposed to most of the body. To obtain widespread in vivo distribution, a blood half-life such that the drug is not rapidly degraded or cleared (e.g., by first-pass organs (liver, lung, etc.) or rapid non-specific cell binding) before reaching a diseased site distal to the administration site is generally required. Systemic delivery of lipid particles can be by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of lipid particles is by intravenous delivery.

[0144] As used herein, "local delivery" refers to the direct delivery of an active or therapeutic agent such as interfering RNA or mRNA to a target site in the body. For example, the drug can be locally delivered by direct injection to a diseased site such as a tumor, or other target sites such as an inflammatory site, or a target organ such as the liver, heart, pancreas, kidney, etc.

[0145] The term "mammal" refers to any mammalian species, such as, for example, humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, domestic animals, etc.

[0146] The term "cancer" refers to any member of a class of diseases characterized by the uncontrolled growth of abnormal cells. This term includes all known cancers and tumor states, whether characterized as malignant, benign, soft tissue or solid, and all stages and grades of cancer, including pre-metastatic and post-metastatic cancer. Examples of different types of cancer include, but are not limited to, lung cancer, colon cancer, rectal cancer, anal cancer, cholangiocarcinoma, 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), cancers of the central nervous system, glioblastoma, skin cancer, lymphoma, choriocarcinoma, head and neck cancer, osteosarcoma, and blood cancers. Non-limiting examples of specific types of liver cancer include hepatocellular carcinoma (HCC), secondary liver cancer (e.g., resulting from metastases of several other non-liver cancer cell types), and hepatoblastoma. As used herein, a "tumor" contains one or more cancer cells.

[0147] The term "anion precursor group" includes groups that can form ions at physiological pH. For example, this term includes the groups -CO2H, -O-P(=O)(OH)2, -OS(=O)2(OH), -O-S(=O)(OH), and -B(OH)2. In one embodiment, the anion precursor is -CO2H.

[0148] In certain embodiments, PEG-C-DMA has the following structure:

Chemical formula

[0149] Description of Embodiments The present invention provides novel serum-stable lipid particles comprising one or more active agents or therapeutic agents, methods of making the lipid particles, and methods of delivering and / or administering the lipid particles (e.g., for the treatment of a disease or disorder).

[0150] In one aspect, the present invention provides lipid particles comprising (a) one or more active agents 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 aggregation of the particles, constituting about 0.1 mol% to about 10 mol% of the total lipids present in the particles.

[0151] In one aspect, the present invention provides lipid particles comprising (a) one or more active agents 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 aggregation of the particles, constituting about 0.5 mol% to about 2 mol% of the total lipids present in the particles.

[0152] In certain embodiments, the active or therapeutic agent is completely encapsulated within the lipid portion of the lipid particle such that the active or therapeutic agent in the lipid particle is resistant to enzymatic degradation, e.g., by nucleases or proteases, in an aqueous solution. In certain other embodiments, the lipid particles are substantially non-toxic to mammals such as humans.

[0153] In some embodiments, the active or therapeutic agent comprises a nucleic acid. In certain cases, the nucleic acid comprises interfering RNA molecules such as, for example, 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, for example, antisense oligonucleotides, ribozymes, plasmids, immunostimulatory oligonucleotides, or mixtures thereof. In certain cases, the nucleic acid comprises an mRNA molecule.

[0154] In other embodiments, the active 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, apoptosis factors, differentiation-inducing factors, cell surface receptors, ligands, hormones, small molecules (e.g., organic small molecules or compounds), or mixtures thereof.

[0155] In one embodiment, the active or therapeutic agent comprises siRNA. In one embodiment, the siRNA molecule comprises a double-stranded region that is about 15 to about 60 nucleotides in length (e.g., about 15 - 60, 15 - 50, 15 - 40, 15 - 30, 15 - 25, or 19 - 25 nucleotides in length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). The siRNA molecules of the present invention can silence the expression of a target sequence in vitro and / or in vivo.

[0156] In some embodiments, the siRNA molecule comprises at least one modified nucleotide. In certain preferred embodiments, the siRNA molecule comprises one, two, three, four, five, six, seven, eight, nine, ten, or more modified nucleotides in the double-stranded region. In certain cases, the siRNA comprises from 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 about 25% (e.g., less than about 25%, less than 20%, less than 15%, less than 10%, or less than 5%) or from about 1% to about 25% (e.g., from about 1% to 25%, from 5% to 25%, from 10% to 25%, from 15% to 25%, from 20% to 25%, or from 10% to 20%) of the nucleotides in the double-stranded region comprise modified nucleotides.

[0157] In other embodiments, the siRNA molecule comprises modified nucleotides including, but not limited to, 2'-O-methyl (2'OMe) nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'-deoxynucleotides, 2'-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof. In a preferred embodiment, the siRNA comprises 2'OMe nucleotides (e.g., 2'OMe purine and / or pyrimidine nucleotides) such as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, 2'OMe-cytosine nucleotides, and mixtures thereof. In certain cases, the siRNA does not comprise 2'OMe-cytosine nucleotides. In other embodiments, the siRNA comprises a hairpin loop structure.

[0158] The siRNA can contain modified nucleotides in 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 selected positions in the double-stranded region of the siRNA duplex. With respect to uridine nucleotide modification, at least 1, 2, 3, 4, 5, 6, or more of the uridine nucleotides in the sense strand and / or antisense strand can be modified uridine nucleotides such as 2’OMe-uridine nucleotides. In some embodiments, all of the uridine nucleotides in the sense strand and / or antisense strand are 2’OMe-uridine nucleotides. With respect to guanosine modification, at least 1, 2, 3, 4, 5, 6, or more of the guanosine nucleotides in the sense strand and / or antisense strand can be modified guanosine nucleotides such as 2’OMe-guanosine nucleotides. In some embodiments, all of the guanosine nucleotides in the sense strand and / or antisense strand are 2’OMe-guanosine nucleotides.

[0159] In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, or more 5’-GU-3’ motifs of the siRNA sequence can be modified, for example, by introducing mismatches to remove the 5’-GU-3’ motif and / or by introducing modified nucleotides such as 2’OMe nucleotides. The 5’-GU-3’ motif can be present in the sense strand, antisense strand, or both strands of the siRNA sequence. The 5’-GU-3’ motifs can be adjacent to each other or alternatively separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides.

[0160] In some preferred embodiments, the modified siRNA molecule is less immunostimulatory than the corresponding unmodified siRNA sequence. In such embodiments, the modified siRNA molecule with reduced immunostimulatory properties favorably retains RNAi activity against the target sequence. In another embodiment, the immunostimulatory properties of the modified siRNA molecule and its ability to silence target gene expression can be balanced or optimized by introducing minimal and selective 2'-OMe modifications within the siRNA sequence, such as within the double-stranded region of the siRNA duplex. In certain cases, the modified siRNA is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% less immunostimulatory than the corresponding unmodified siRNA. It will be readily apparent to those skilled in the art that the immunostimulatory properties of the modified siRNA molecule and the corresponding unmodified siRNA molecule can be determined, for example, by measuring INF-α and / or IL-6 levels in mammals at about 2 hours to about 12 hours after systemic administration in mammals or transfection of responder cells of mammals using a suitable lipid-based delivery system (such as the LNP delivery system disclosed herein).

[0161] In certain embodiments, the modified siRNA molecule has an IC 50 (i.e., the maximum half-inhibitory concentration) that is 10-fold or less of the IC 50 of the corresponding unmodified siRNA (i.e., the modified siRNA has an IC 50 that is 10-fold or less of the IC 50 of the corresponding unmodified siRNA). In other embodiments, the modified siRNA has an IC 50 that is 3-fold or less of the IC 50 of the corresponding unmodified siRNA sequence. In yet other embodiments, the modified siRNA has an IC 50 that is 2-fold or less of the IC 50 of the corresponding unmodified siRNA. Dose-response curves can be generated using methods well known to those skilled in the art, and the IC50 It will be readily apparent to those skilled in the art that the value can be easily measured.

[0162] 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 as compared to the corresponding unmodified siRNA.

[0163] In some embodiments, the siRNA molecule does not contain a phosphate backbone modification, for example, in the sense strand and / or the antisense strand 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 strand and / or the antisense strand of the double-stranded region. In a preferred embodiment, the siRNA does not contain a phosphate backbone modification.

[0164] In a further embodiment, the siRNA does not contain 2'-deoxynucleotides, for example, in the sense strand and / or the antisense strand of the double-stranded region. In yet a further embodiment, the siRNA contains one, two, three, four, or more 2'-deoxynucleotides, for example, in the sense strand and / or the antisense strand of the double-stranded region. In a preferred embodiment, the siRNA does not contain 2'-deoxynucleotides.

[0165] In certain cases, the nucleotides at the 3' end of the double-stranded region of the sense strand and / or the antisense strand are not modified nucleotides. In certain other cases, the nucleotides near the 3' end (e.g., within 1, 2, 3, or 4 nucleotides of the 3' end) of the double-stranded region of the sense strand and / or the antisense strand are not modified nucleotides.

[0166] The siRNA molecules described herein may have 3' overhangs of one, two, three, four or more nucleotides on one or both sides of the double-stranded region, or may lack overhangs on one or both sides of the double-stranded region (i.e., may have blunt ends). Preferably, the siRNA has 3' overhangs of two nucleotides on both sides of the double-stranded region. In certain cases, the 3' overhang on the antisense strand has complementarity to the target sequence, and the 3' overhang on the sense strand has complementarity to the complementary strand of the target sequence. Alternatively, the 3' overhang does not have complementarity to the target sequence or its complementary strand. In some embodiments, the 3' overhang contains one, two, three, four, or more nucleotides such as 2'-deoxy (2'H) nucleotides. In certain preferred embodiments, the 3' overhang contains deoxythymidine (dT) and / or uridine nucleotides. In other embodiments, one or more of the nucleotides in the 3' overhang on one or both sides of the double-stranded region contain modified nucleotides. Non-limiting examples of modified nucleotides have been described above and include 2'OMe nucleotides, 2'-deoxy-2'F nucleotides, 2'-deoxynucleotides, 2'-O-2-MOE nucleotides, LNA nucleotides, and mixtures thereof. In preferred embodiments, one, two, three, four, or more of the nucleotides in the 3' overhang present on the sense strand and / or antisense strand of the siRNA include, for example, 2'OMe nucleotides such as 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, 2'OMe-cytosine nucleotides (e.g., 2'OMe purine and / or pyrimidine nucleotides), and mixtures thereof.

[0167] The siRNA can comprise at least one or a cocktail of unmodified and / or modified siRNA sequences (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) that silence the expression of a target gene. The cocktail of siRNAs can comprise sequences targeting the same region or domain (e.g., a “hot spot”) and / or different regions or domains of one or more target genes. In certain cases, modified siRNAs that silence the expression of one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) target genes are present in the cocktail. In certain other cases, unmodified siRNA sequences that silence the expression of one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) target genes are present in the cocktail.

[0168] In some embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence or a portion thereof. In other embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that is 100% complementary to the target sequence or a portion thereof. In further embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that specifically hybridizes to the target sequence or a portion thereof.

[0169] In further embodiments, the sense strand of the siRNA molecule comprises or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the target sequence or a portion thereof. In additional embodiments, the sense strand of the siRNA molecule comprises or consists of a sequence that is 100% identical to the target sequence or a portion thereof.

[0170] In the lipid nanoparticles of the present invention, the cationic lipid can be selected from the compounds of formula (I) described herein.

[0171] In some embodiments, the cationic lipid may constitute from about 30 mol% to about 90 mol%, from about 30 mol% to about 85 mol%, from about 30 mol% to about 80 mol%, from about 30 mol% to about 75 mol%, from about 30 mol% to about 70 mol%, from about 30 mol% to about 65 mol%, or from about 30 mol% to about 60 mol% of the total lipids present in the particles.

[0172] In some embodiments, the cationic lipid may constitute from about 40 mol% to about 90 mol%, from about 40 mol% to about 85 mol%, from about 40 mol% to about 80 mol%, from about 40 mol% to about 75 mol%, from about 40 mol% to about 70 mol%, from about 40 mol% to about 65 mol%, or from about 40 mol% to about 60 mol% of the total lipids present in the particles.

[0173] In other embodiments, the cationic lipid may constitute from about 55 mol% to about 90 mol%, from about 55 mol% to about 85 mol%, from about 55 mol% to about 80 mol%, from about 55 mol% to about 75 mol%, from about 55 mol% to about 70 mol%, or from about 55 mol% to about 65 mol% of the total lipids present in the particles.

[0174] In still other embodiments, the cationic lipid may constitute from about 60 mol% to about 90 mol%, from about 60 mol% to about 85 mol%, from about 60 mol% to about 80 mol%, from about 60 mol% to about 75 mol%, or from about 60 mol% to about 70 mol% of the total lipids present in the particles.

[0175] In yet further other embodiments, the cationic lipid may constitute from about 65 mol% to about 90 mol%, from about 65 mol% to about 85 mol%, from about 65 mol% to about 80 mol%, or from about 65 mol% to about 75 mol% of the total lipids present in the particles.

[0176] In a further embodiment, the cationic lipid can 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.

[0177] In additional embodiments, the cationic lipid can 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 or range therebetween) of the total lipids present in the particles.

[0178] In the lipid particles of the present invention, the non-cationic lipid can include, for example, one or more anionic lipids and / or neutral lipids. In a preferred embodiment, the non-cationic lipid includes one of the following neutral lipid components: (1) cholesterol or a derivative thereof, (2) a phospholipid, or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.

[0179] 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.

[0180] The phospholipids can be neutral lipids including, but not limited to, dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl-phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), palmitoyl oleoyl-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyl oleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), and mixtures thereof. In certain preferred embodiments, the phospholipid is DPPC, DSPC, or a mixture thereof.

[0181] In some embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) can 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. When the non-cationic lipid is a mixture of a phospholipid and cholesterol or a cholesterol derivative, the mixture can constitute up to about 40, 50, or 60 mol% of the total lipids present in the particles.

[0182] In other embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) can constitute from about 10 mol% to about 49.5 mol%, from about 13 mol% to about 49.5 mol%, from about 15 mol% to about 49.5 mol%, from about 20 mol% to about 49.5 mol%, from about 25 mol% to about 49.5 mol%, from about 30 mol% to about 49.5 mol%, from about 35 mol% to about 49.5 mol%, or from about 40 mol% to about 49.5 mol% of the total lipids present in the particles.

[0183] In yet other embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) can constitute from about 10 mol% to about 45 mol%, from about 13 mol% to about 45 mol%, from about 15 mol% to about 45 mol%, from about 20 mol% to about 45 mol%, from about 25 mol% to about 45 mol%, from about 30 mol% to about 45 mol%, or from about 35 mol% to about 45 mol% of the total lipids present in the particles.

[0184] In still further other embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) can constitute from about 10 mol% to about 40 mol%, from about 13 mol% to about 40 mol%, from about 15 mol% to about 40 mol%, from about 20 mol% to about 40 mol%, from about 25 mol% to about 40 mol%, or from about 30 mol% to about 40 mol% of the total lipids present in the particles.

[0185] In further embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) can constitute from about 10 mol% to about 35 mol%, from about 13 mol% to about 35 mol%, from about 15 mol% to about 35 mol%, from about 20 mol% to about 35 mol%, or from about 25 mol% to about 35 mol% of the total lipids present in the particles.

[0186] In yet further embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) can 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.

[0187] In additional embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) can 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 or range therebetween) of the total lipids present in the particles.

[0188] In certain preferred embodiments, the non-cationic lipid comprises cholesterol or a derivative thereof at about 31.5 mol% to about 42.5 mol% of the total lipids present in the particles. By way of non-limiting example, the lipid-free particles of the invention can comprise cholesterol or a derivative thereof at about 37 mol% of the total lipids present in the particles. In other preferred embodiments, the lipid-free particles of the invention can comprise cholesterol or a derivative thereof at 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 or range therebetween) of the total lipids present in the particles.

[0189] In certain other preferred embodiments, the non-cationic lipid comprises a mixture of (i) phospholipid at about 4 mol% to about 10 mol% of the total lipid present in the particle and (ii) cholesterol or a derivative thereof at about 30 mol% to about 40 mol% of the total lipid present in the particle. As a non-limiting example, lipid particles comprising a mixture of phospholipid and cholesterol can comprise about 7 mol% DPPC and about 34 mol% cholesterol of the total lipid present in the particle. In other embodiments, the non-cationic lipid comprises a mixture of (i) phospholipid at 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 or range therein) of the total lipid present in the particle and (ii) cholesterol or a derivative thereof at about 25 mol% to about 45 mol%, about 30 mol% to about 45 mol%, about 25 mol% to about 40 mol%, about 30 mol% to about 40 mol%, about 25 mol% to about 35 mol%, about 30 mol% to about 35 mol%, about 35 mol% to about 45 mol%, about 40 mol% to about 45 mol%, about 28 mol% to about 40 mol%, about 28 mol% to about 38 mol%, about 30 mol% to about 38 mol%, about 32 mol% to about 36 mol%, or about 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 or range therein) of the total lipid present in the particle.

[0190] In a more preferred embodiment, the non-cationic lipid comprises a mixture of (i) phospholipids at about 10 mol% to about 30 mol% of the total lipids present in the particles and (ii) cholesterol or a derivative thereof at about 10 mol% to about 30 mol% of the total lipids present in the particles. As a non-limiting example, lipid particles comprising a mixture of phospholipid and cholesterol may comprise about 20 mol% DPPC and about 20 mol% cholesterol of the total lipids present in the particles. In other embodiments, the non-cationic lipid is (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 or range therein) of phospholipids of the total lipids present in the particles and (ii) 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 or range therein) of cholesterol or a derivative thereof of the total lipids present in the particles.

[0191] Conjugated lipid In the lipid particles of the present invention (e.g., LNP (including interfering RNAs such as siRNA, or mRNA)), the conjugate lipid can include, for example, one or more of the following, namely polyethylene glycol (PEG)-lipid conjugate, polyamide (ATTA)-lipid conjugate, or a mixture thereof. In a preferred embodiment, the nucleic acid-lipid particle contains either a PEG-lipid conjugate or an ATTA-lipid conjugate. The conjugate lipid can include, for example, PEG-lipids including PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be PEG-dilauroxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmityloxypropyl (C16), PEG-distearyloxypropyl (C18), or a mixture thereof.

[0192] 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 No. PCT / US08 / 88676 filed on December 31, 2008, and this disclosure is hereby incorporated by reference in its entirety for all purposes. Even further PEG-lipid conjugates suitable for use in the present invention include, but are not limited to, 1-[8’-(1,2-dimyristoyl-3-propaneoxy)-carboxamido-3’,6’-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethylene glycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S. Patent No. 7,404,969, and this disclosure is hereby incorporated by reference in its entirety for all purposes.

[0193] The PEG moiety of the PEG-lipid conjugate described herein can have an average molecular weight in the range of about 550 Daltons to about 10,000 Daltons. In certain cases, the PEG moiety 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 a preferred embodiment, the PEG moiety has an average molecular weight of about 2,000 Daltons or about 750 Daltons.

[0194] In certain cases, the conjugate lipid (e.g., PEG-lipid conjugate) can constitute about 0.1 to about 10% (or any fraction or range therein) of the total lipid present in the particles. In certain cases, the conjugate lipid (e.g., PEG-lipid conjugate) can constitute about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol%, about 1 mol% to about 2 mol%, about 0.6 mol% to about 1.9 mol%, about 0.7 mol% to about 1.8 mol%, about 0.8 mol% to about 1.7 mol%, about 1 mol% to about 1.8 mol%, about 1.2 mol% to about 1.8 mol%, about 1.2 mol% to about 1.7 mol%, about 1.3 mol% to about 1.6 mol%, about 1.4 mol% to about 1.5 mol%, or about 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 or range therein) of the total lipid present in the particles.

[0195] In the lipid particles of the present invention, the active agent or therapeutic agent is completely encapsulated within the lipid portion of the particle, thereby protecting the active agent or therapeutic agent from nuclease degradation. In a preferred embodiment, an LNP containing a nucleic acid such as an interfering RNA (e.g., siRNA) or mRNA is 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 is not substantially degraded after exposing the particles to nuclease at 37 °C for at least about 20, 30, 45, or 60 minutes. In certain other cases, the nucleic acid in the LNP is not substantially degraded after incubating the particles in serum at 37 °C for at least about 30, 45, or 60 minutes or for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the active agent 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.

[0196] The term "fully encapsulated" indicates that the active or therapeutic agent in the lipid particle is not significantly degraded after exposure to serum or nuclease or protease assays that can significantly degrade free DNA, RNA, or protein. In a fully encapsulated system, in a process that normally degrades 100% of the free active or therapeutic agent, preferably less than about 25% of the active or therapeutic agent in the particle is degraded, more preferably less than about 10% of the active or therapeutic agent in the particle, and most preferably less than about 5% of the active or therapeutic agent in the particle is degraded. In the context of nucleic acid therapeutics, complete encapsulation can be determined by the Oligreen® assay. Oligreen® is a highly sensitive fluorescent nucleic acid stain for quantifying oligonucleotides and single-stranded DNA or RNA in solution (available from Invitrogen Corporation; Carlsbad, Calif.). "Fully encapsulated" also indicates that the lipid particles are serum stable, i.e., they are not rapidly degraded into their components upon in vivo administration.

[0197] In another aspect, the present invention provides a lipid particle (e.g., LNP) composition comprising a plurality of lipid particles. In preferred embodiments, the active or therapeutic agent (e.g., nucleic acid) is fully encapsulated within the lipid portion of the lipid particle (e.g., LNP) such that 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%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any fraction or range therebetween) of the active or therapeutic agent is encapsulated therein.

[0198] Typically, the lipid particles (e.g., LNP) of the present invention have a lipid:agent (e.g., lipid:nucleic acid) ratio (mass / mass ratio) of about 1 to about 100. In some cases, the lipid:agent (e.g., lipid:nucleic acid) ratio (mass / mass ratio) ranges from 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 a preferred embodiment, the lipid particles of the present invention have a lipid:agent (e.g., lipid:nucleic acid) ratio (mass / mass ratio) of about 5 to about 15, such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (or any fraction or range therebetween).

[0199] Typically, the lipid particles (e.g., LNP) 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., LNP) 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 or range therebetween).

[0200] 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 a target gene such as siRNA, aiRNA, miRNA, or mRNA that results in the expression of a target protein), (b) a cationic lipid that constitutes from about 56.5 mol% to about 66.5 mol% of the total lipids present in the particle, (c) a non-cationic lipid that constitutes from about 31.5 mol% to about 42.5 mol% of the total lipids present in the particle, and (d) a conjugate lipid that inhibits aggregation of the particle and constitutes from about 1 mol% to about 2 mol% of the total lipids present in the particle. This particular embodiment of the LNP is generally referred to herein as the "1:62" formulation. In a preferred embodiment, 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.

[0201] 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 mRNAs that give rise to the expression of target proteins), (b) a cationic lipid that constitutes about 52 mol% to about 62 mol% of the total lipids present in the particle, (c) a non-cationic lipid that constitutes about 36 mol% to about 47 mol% of the total lipids present in the particle, and (d) a conjugate lipid that inhibits aggregation of the particles and constitutes about 1 mol% to about 2 mol% of the total lipids present in the particle. This specific embodiment of the LNP is generally referred to herein as the "1:57" formulation. In a preferred embodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA ("XTC2"), the non-cationic lipid is a mixture of a phospholipid (such as DPPC) and cholesterol (where the phospholipid constitutes about 5 mol% to about 9 mol% (e.g., about 7.1 mol%) of the total lipids present in the particle and cholesterol (or a cholesterol derivative) constitutes about 32 mol% to about 37 mol% (e.g., about 34.3 mol%) of the total lipids present in the particle), 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 a 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 particle and cholesterol (or a cholesterol derivative) constitutes about 15 mol% to about 25 mol% (e.g., about 20 mol%) of the total lipids present in the particle), 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.

[0202] In a preferred embodiment, the 1:62 LNP formulation is a three-component system that does not contain phospholipids and is composed of approximately 1.5 mol% PEG-cDMA (or PEG-IDSA), approximately 61.5 mol% DLinDMA (or XTC2), and approximately 36.9 mol% cholesterol (or its derivative). In another preferred embodiment, the 1:57 LNP formulation is a four-component system composed of approximately 1.4 mol% PEG-cDMA (or PEG-cDSA), approximately 57.1 mol% DLinDMA (or XTC2), approximately 7.1 mol% DPPC, and approximately 34.3 mol% cholesterol (or its derivative). In yet another preferred embodiment, the 1:57 LNP formulation is a four-component system composed of approximately 1.4 mol% PEG-cDMA (or PEG-cDSA), approximately 57.1 mol% DLinDMA (or XTC2), approximately 20 mol% DPPC, and approximately 20 mol% cholesterol (or its derivative). These LNP formulations are the target formulations, and it should be understood that the amounts of lipids (both cationic and non-cationic) present in the LNP formulation and the amounts of lipid conjugates present can vary.

[0203] The present invention also provides a pharmaceutical composition comprising the lipid particles (e.g., LNP) described herein and a pharmaceutically acceptable carrier.

[0204] In a further aspect, the present invention provides a method for introducing one or more active agents or therapeutic agents (e.g., nucleic acids) into cells, the method comprising contacting the cells with the lipid particles (e.g., LNP) 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 active agents or therapeutic agents (e.g., nucleic acids), the method comprising administering the lipid particles (e.g., LNP) described herein to a mammalian subject. In preferred embodiments, the modes of administration include, but are not limited to, oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal. Preferably, the mammalian subject is a human.

[0205] In one embodiment, at least about 5%, 10%, 15%, 20%, or 25% of the total injected dose of lipid particles (e.g., LNP) is present in 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 up to about 60%, 70%, or 80% of the total injected dose of lipid particles (e.g., LNP) is present in plasma about 8, 12, 24, 36, or 48 hours after injection. In certain cases, more than about 10% of a plurality of particles is present in the plasma of a mammal about 1 hour after administration. In certain other cases, the presence of lipid particles (e.g., LNP) is detectable at least about 1 hour after administration of the particles. In certain embodiments, the presence of an active agent or therapeutic agent such as interfering RNA (e.g., siRNA) or mRNA is detectable intracellularly (e.g., in the lung, liver, tumor, or site of inflammation) about 8, 12, 24, 36, 48, 60, 72, or 96 hours after administration. In other embodiments, downregulation of the expression of a target sequence by an active agent 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 still other embodiments, downregulation of the expression of a target sequence by an active agent or therapeutic agent such as interfering RNA (e.g., siRNA) preferentially occurs within tumor cells or within cells at the site of inflammation. In further embodiments, the presence or action of an active agent or therapeutic agent such as interfering RNA (e.g., siRNA) intracellularly at a site proximal or distal to the site of administration or within cells of the lung, liver, or tumor is detectable about 12, 24, 48, 72, or 96 hours after administration, or about 6, 8, 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days after administration. In other embodiments, upregulation of the expression of a target sequence by an active agent or therapeutic agent such as mRNA or self-amplifying RNA is detectable about 8, 12, 24, 36, 48, 60, 72, or 96 hours after administration. In still other embodiments, upregulation of the expression of a target sequence by an active agent or therapeutic agent such as mRNA or self-amplifying RNA preferentially occurs within tumor cells or within cells at the site of inflammation.In further embodiments, the presence or action of an active or therapeutic agent, such as mRNA or self-replicating RNA, within cells at a site proximal or distal to the administration site or within cells of the lung, liver, or tumor, is detectable about 12, 24, 48, 72, or 96 hours after administration, or about 6, 8, 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days after administration. In additional embodiments, the lipid particles (e.g., LNPs) of the invention are administered parenterally or intraperitoneally.

[0206] In some embodiments, the lipid particles (e.g., LNPs) of the invention are particularly useful for methods for the therapeutic delivery of one or more nucleic acids, including interfering RNA sequences (e.g., siRNA). In particular, it is an object of the invention to provide in vitro and in vivo methods for treating a disease or disorder in a mammal (e.g., a rodent such as a mouse or a primate such as a human, chimpanzee, or monkey) by downregulating or silencing the transcription and / or translation of one or more target nucleic acid sequences or genes of interest. By way of non-limiting example, the methods of the invention are useful for the in vivo delivery of interfering RNA (e.g., siRNA) to the liver and / or tumors of a mammalian subject. In certain embodiments, the disease or disorder is associated with the expression and / or overexpression of a gene, and the expression or overexpression of the gene 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. Optionally, interfering RNA (e.g., siRNA) is formulated into LNPs and these particles are administered to a patient in need of such treatment. In other cases, cells are removed from the patient, interfering RNA (e.g., siRNA) is delivered in vitro (e.g., using LNPs described herein), and the cells are reinjected into the patient.

[0207] In an additional aspect, the invention provides lipid particles (e.g., LNPs) comprising an asymmetric interfering RNA (aiRNA) molecule that silences the expression of a target gene, and methods of using such particles to silence the expression of a target gene.

[0208] In one embodiment, the aiRNA molecule comprises a double-stranded region that is about 10 to about 25 (base-pairing) nucleotides in length, the aiRNA molecule comprises an antisense strand that includes 5' and 3' overhangs, and the aiRNA molecule can silence target gene expression.

[0209] In one embodiment, the aiRNA molecule comprises a double-stranded region that is about 12 to 20, 12 to 19, 12 to 18, 13 to 17, or 14 to 17 (base-pairing) nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 (base-pairing) nucleotides in length. In certain other cases, the 5' and 3' overhangs on the antisense strand include sequences that are complementary to the target RNA sequence and optionally may further include non-target sequences. In some embodiments, each of the 5' and 3' overhangs on the antisense strand comprises, or consists of, 1, 2, 3, 4, 5, 6, 7, or more nucleotides.

[0210] In other embodiments, the aiRNA molecule comprises modified nucleotides selected from the group consisting of 2'OMe nucleotides, 2'F nucleotides, 2'-deoxynucleotides, 2'-O-MOE nucleotides, LNA nucleotides, and mixtures thereof. In a preferred embodiment, the aiRNA molecule comprises 2'OMe nucleotides. By way of non-limiting example, the 2'OMe nucleotides can be selected from the group consisting of 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, and mixtures thereof.

[0211] In related aspects, the invention provides lipid particles (e.g., LNPs) comprising microRNA (miRNA) molecules that silence the expression of a target gene, and methods of using such compositions to silence target gene expression.

[0212] In one embodiment, the miRNA molecule is composed of about 15 to about 60 nucleotides in length and the miRNA molecule can silence the expression of a target gene.

[0213] In certain cases, the miRNA molecule is composed of about 15 to 50, 15 to 40, or 15 to 30 nucleotides in length, more typically about 15 to 25 or 19 to 25 nucleotides in length, preferably about 20 to 24, 21 to 22, or 21 to 23 nucleotides in length. In a preferred embodiment, the miRNA molecule is a mature miRNA molecule that targets a target RNA sequence.

[0214] In some embodiments, the miRNA 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 a preferred embodiment, the miRNA molecule comprises a 2′-OMe nucleotide. By way of non-limiting example, the 2′-OMe nucleotide can be selected from the group consisting of 2′-OMe-guanosine nucleotides, 2′-OMe-uridine nucleotides, and mixtures thereof.

[0215] In some embodiments, the lipid particles (e.g., LNPs) of the present invention are useful in methods for the therapeutic delivery of one or more mRNA molecules. In particular, it is an object of the present invention to provide in vitro and in vivo methods for treating a disease or disorder in a mammal (e.g., a rodent such as a mouse or a primate such as a human, chimpanzee, or monkey) via the expression of one or more target proteins. By way of non-limiting example, the methods of the present invention are useful for the in vivo delivery of one or more mRNA molecules to a mammalian subject. 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 the particles are administered to a patient in need of such treatment. In other cases, cells are removed from a patient, one or more mRNA molecules are delivered in vitro (e.g., using LNPs described herein), and the cells are reinjected into the patient.

[0216] In other embodiments, the mRNA molecule comprises modified nucleotides 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 aspects, the invention provides lipid particles (e.g., LNPs) comprising microRNA (miRNA) molecules that silence the expression of a target gene, and methods of using such compositions to silence target gene expression.

[0217] Accordingly, the lipid particles (e.g., LNPs) of the invention are of a size required for their pharmacodynamic behavior such that they are stable in circulation and provide access to extravascular sites and can reach the target cell population, and are thus convenient and suitable for use in the administration of an active agent or therapeutic agent, such as a nucleic acid (e.g., an interfering RNA such as siRNA, aiRNA, and / or miRNA, or mRNA), to a subject (e.g., a mammal such as a human).

[0218] Active agent The active agent (e.g., therapeutic agent) includes any molecule or compound that can exert a desired effect on cells, tissues, organs, or a subject. Such effects can be, for example, biological, physiological, and / or cosmetic effects. The active agent can be any type of molecule or compound, including but not limited to nucleic acids, peptides, polypeptides, small molecules, and mixtures thereof. Non-limiting examples of nucleic acids include interfering RNA molecules (e.g., siRNA, aiRNA, miRNA), antisense oligonucleotides, mRNA, self-amplifying RNA, plasmids, ribozymes, immunostimulatory oligonucleotides, and mixtures thereof. Examples of peptides or polypeptides include antibodies (e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments; humanized antibodies, recombinant antibodies, recombinant human antibodies, Primatized™ antibodies), cytokines, growth factors, apoptosis factors, differentiation-inducing factors, cell surface receptors and their ligands, hormones, and mixtures thereof, but are not limited thereto. Examples of small molecules include, but are not limited to, organic small molecules or compounds such as any conventional drug or medicine known to those skilled in the art.

[0219] In some embodiments, the active agent is a therapeutic agent, or a salt or derivative thereof. The therapeutic agent derivative may itself have therapeutic activity or may be a prodrug that becomes active upon further modification. Thus, in one embodiment, the therapeutic agent derivative retains some or all of the therapeutic activity compared to the unmodified drug, while in another embodiment, the therapeutic agent derivative is a prodrug that lacks therapeutic activity but becomes active upon further modification.

[0220] Nucleic acid In certain embodiments, the lipid particles of the invention associate with a nucleic acid to form a nucleic acid-lipid particle (e.g., LNP). In some embodiments, the nucleic acid is completely encapsulated within the lipid particle. As used herein, the term "nucleic acid" generally 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 invention are from about 15 to about 60 nucleotides in length. The nucleic acid can be administered alone with the lipid particles of the invention or in combination (e.g., co-administered) with the lipid particles of the invention that contain small molecules such as peptides, polypeptides, or conventional drugs.

[0221] In the context of the present invention, the terms "polynucleotide" and "oligonucleotide" refer to polymers or oligomers of nucleotides or nucleoside monomers consisting of natural bases, sugars, and intersugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers that include non-natural monomers or portions thereof that function similarly. Such modified or substituted oligonucleotides are often preferred over their native counterparts due to properties such as improved cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0222] Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleotides. Deoxyribooligonucleotides consist of a five-carbon sugar called deoxyribose, which covalently bonds to phosphate at the 5' and 3' carbons of the sugar to form an alternating unbranched polymer. Ribooligonucleotides consist of a similar repeating structure where the five-carbon sugar is ribose.

[0223] The nucleic acid present in the lipid-nucleic acid particle according to the present invention includes any known form of nucleic acid. The nucleic acid used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or a DNA-RNA hybrid. 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 miRNA, and triplex-forming oligonucleotides.

[0224] The nucleic acid of the present invention can generally be of various lengths depending on the particular form of the nucleic acid. For example, in certain embodiments, a plasmid or gene can be about 1,000 to about 100,000 nucleotide residues in length. In certain embodiments, an oligonucleotide can be in the range of about 10 to about 100 nucleotides in length. In various related embodiments, oligonucleotides, whether single-stranded, double-stranded, or triple-stranded, can be in the range of about 10 to about 60 nucleotides in length, about 15 to about 60 nucleotides in length, about 20 to about 50 nucleotides in length, about 15 to about 30 nucleotides in length, or about 20 to about 30 nucleotides in length.

[0225] In certain embodiments, the oligonucleotides (or strands thereof) of the invention specifically hybridize to, or are complementary to, a target polynucleotide sequence. As used herein, the terms "specifically hybridizable" and "complementary" indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It should be understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. In preferred embodiments, an oligonucleotide is specifically hybridizable when its binding to the target sequence interferes with the normal function of the target sequence to cause a loss of utility or expression from the target sequence, and has a sufficient degree of complementarity to avoid non-specific binding to non-target sequences of the oligonucleotide under conditions where specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatments, or under the conditions under which the assay is performed in the case of in vitro assays. Thus, an oligonucleotide can contain one, two, three, or more base substitutions compared to the region of the gene or mRNA sequence to which it targets or with which it specifically hybridizes.

[0226] 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 duplex is usually about 15 to about 60 nucleotides in length, preferably about 15 to about 30 nucleotides in length. In certain embodiments, the siRNA contains at least one modified nucleotide. Modified siRNAs are generally less immunostimulatory than their corresponding unmodified siRNA sequences and retain RNAi activity against the target gene of interest. In some embodiments, the modified siRNA contains at least one 2′-OMe purine nucleotide or 2′-OMe pyrimidine nucleotide, such as a 2′-OMe-guanosine nucleotide, 2′-OMe-uridine nucleotide, 2′-OMe-adenosine nucleotide, and / or 2′-OMe-cytosine nucleotide. In a preferred embodiment, one or more of the uridine and / or guanosine nucleotides are modified. The modified nucleotides can be present in one strand (i.e., sense or antisense) or both strands of the siRNA. The siRNA sequence may have 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., have blunt ends).

[0227] Modified siRNAs generally contain from about 1% to about 100% (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in the double-stranded region of the siRNA duplex. In certain embodiments, one, two, three, four, five, six, seven, eight, nine, ten, or more of the nucleotides within the double-stranded region of the siRNA contain modified nucleotides.

[0228] In some embodiments, less than about 25% (e.g., less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%) of the nucleotides within the double-stranded region of the siRNA comprise modified nucleotides.

[0229] In other embodiments, about 1% to about 25% (e.g., about 1% - 25%, 2% - 25%, 3% - 25%, 4% - 25%, 5% - 25%, 6% - 25%, 7% - 25%, 8% - 25%, 9% - 25%, 10% - 25%, 11% - 25%, 12% - 25%, 13% - 25%, 14% - 25%, 15% - 25%, 16% - 25%, 17% - 25%, 18% - 25%, 19% - 25%, 20% - 25%, 21% - 25%, 22% - 25%, 23% - 25%, 24% - 25%, etc.) or about 1% to about 20% (e.g., about 1% - 20%, 2% - 20%, 3% - 20%, 4% - 20%, 5% - 20%, 6% - 20%, 7% - 20%, 8% - 20%, 9% - 20%, 10% - 20%, 11% - 20%, 12% - 20%, 13% - 20%, 14% - 20%, 15% - 20%, 16% - 20%, 17% - 20%, 18% - 20%, 19% - 20%, 1% - 19%, 2% - 19%, 3% - 19%, 4% - 19%, 5% - 19%, 6% - 19%, 7% - 19%, 8% - 19%, 9% - 19%, 10% - 19%, 11% - 19%, 12% - 19%, 13% - 19%, 14% - 19%, 15% - 19%, 16% - 19%, 17% - 19%, 18% - 19%, 1% - 18%, 2% - 18%, 3% - 18%, 4% - 18%, 5% - 18%, 6% - 18%, 7% - 18%, 8% - 18%, 9% - 18%, 10% - 18%, 11% - 18%, 12% - 18%, 13% - 18%, 14% - 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% - 17%, 15% - 17%, 16% - 17%, 1% - 16%, 2% - 16%, 3% - 16%, 4% - 16%, 5% - 16%, 6% - 16%, 7% - 16%, 8% - 16%, 9% - 16%, 10% - 16%, 11% - 16%, 12% - 16%, 13% - 16%, 14% - 16%, 15% - 16%, 1% - 15%, 2% - 15%, 3% - 15%, 4% - 15%, 5% - 15%, 6% - 15%, 7% - 15%, 8% - 15%, 9% - 15%, 10% - 15%, 11% - 15%, 12% - 15%, 13% - 15%, 14% - 15%, etc.) of the nucleotides within the double-stranded region of the siRNA comprise modified nucleotides.

[0230] In a further embodiment, for example, when one or both strands of the siRNA are selectively modified in uridine and / or guanosine nucleotides, the resulting modified siRNA has less than about 30% modified nucleotides (e.g., less than about 30%, less than about 29%, less than about 28%, less than about 27%, less than about 26%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% modified nucleotides), or from about 1% to about 30% modified nucleotides (e.g., from about 1% to 30%, 2% to 30%, 3% to 30%, 4% to 30%, 5% to 30%, 6% to 30%, 7% to 30%, 8% to 30%, 9% to 30%, 10% to 30%, 11% to 30%, 12% to 30%, 13% to 30%, 14% to 30%, 15% to 30%, 16% to 30%, 17% to 30%, 18% to 30%, 19% to 30%, 20% to 30%, 21% to 30%, 22% to 30%, 23% to 30%, 24% to 30%, 25% to 30%, 26% to 30%, 27% to 30%, 28% to 30%, or 29% to 30% modified nucleotides).

[0231] 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).

[0232] Generally, the 3' nucleotide sequence of the AUG start codon of the transcript from the target gene of interest is scanned for a dinucleotide sequence (e.g., AA, NA, CC, GG, or UU (N = C, G, or U)) (see, for example, Elbashir et al., EMBO J., 20:6877-6888 (2001)). The nucleotide immediately 3' to the dinucleotide sequence is identified as a potential siRNA sequence (e.g., the target sequence or sense strand sequence). Typically, 19, 21, 23, 25, 27, 29, 31, 33, 35, or more nucleotides immediately 3' to the dinucleotide sequence are identified as potential siRNA sequences. In some embodiments, the dinucleotide sequence is an AA or NA sequence, and 19 nucleotides immediately 3' to the AA or NA dinucleotide are identified as a potential siRNA sequence. The siRNA sequences are generally spaced at different positions along the length of the target gene. To further enhance the silencing efficiency of the siRNA sequences, potential siRNA sequences can be analyzed to identify, for example, sites in the target cell or organism that do not contain homologous regions with other coding sequences. For example, a suitable siRNA sequence of about 21 base pairs usually does not have more than 16-17 contiguous base pairs that are homologous to the coding sequence in the target cell or organism. When expressing the siRNA sequence from an RNA PolIII promoter, an siRNA sequence lacking more than four consecutive As or Ts is selected.

[0233] 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 enhance their silencing efficiency, siRNA sequences are analyzed by rational design algorithms, and sequences having one or more of the following characteristics, namely, (1) a G / C content of about 25% to about 60%, (2) at least three A / U at positions 15 to 19 of the sense strand, (3) no internal repeats, (4) position 19 of the sense strand is A, (5) position 3 of the sense strand is A, (6) position 10 of the sense strand is U, (7) position 19 of the sense strand is not G / C, and (8) position 13 of the sense strand is not G, can be identified. siRNA design tools that assign appropriate values to each of these characteristics and incorporate algorithms useful for siRNA selection 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 foregoing characteristics can be selected for further analysis and testing as potential siRNA sequences.

[0234] Furthermore, potential siRNA sequences having one or more of the following criteria, namely, (1) sequences containing four or more consecutive identical bases, (2) sequences containing G homopolymers (e.g., to reduce possible non-specific effects due to the structural characteristics 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 direct repeats of four or more bases within the candidate and resulting in an internal fold-back structure, are often excluded as siRNAs. However, those skilled in the art will understand that sequences having one or more of the foregoing characteristics can still be selected for further analysis and testing as potential siRNA sequences.

[0235] In some embodiments, potential siRNA sequences can be further analyzed based on, for example, the asymmetry of the siRNA duplex as described 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, for example, the secondary structure at the target site as described in Luo et al., Biophys. Res. Commun., 318:303-310 (2004). For example, the 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), and siRNA sequences that are favorable for accessibility at the target site with less secondary structure in the form of base pairing and the presence of few stem loops can be selected.

[0236] After identifying potential siRNA sequences, the sequences can be analyzed for the presence of any immunostimulatory properties, for example, using an in vitro cytokine assay or an in vivo animal model. 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 an indication as to whether the sequence may be immunostimulatory. If it is determined that the siRNA molecule is immunostimulatory, the siRNA molecule can then be modified to reduce its immunostimulatory properties as described herein. As a non-limiting example, to determine whether the siRNA is an immunostimulatory siRNA or a non-immunostimulatory siRNA, the siRNA sequence can be contacted with mammalian responder cells under conditions such that the cells generate a detectable immune response. The mammalian responder cells can be derived from naive mammals (i.e., mammals that have not previously been exposed to the gene product of the siRNA sequence). The mammalian responder cells can be, for example, peripheral blood mononuclear cells (PBMCs), macrophages, etc. The detectable immune response can include, for example, the production of cytokines or growth factors such as TNF-α, IFN-α, IFN-β, IFN-γ, IL-6, IL-12, and combinations thereof. The siRNA molecule identified as immunostimulatory can then be modified by replacing at least one of the nucleotides in the sense and / or antisense strands with a modified nucleotide to reduce its immunostimulatory properties. For example, less than about 30% (e.g., less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%) of the nucleotides within the duplex region of the siRNA duplex can be replaced with a modified nucleotide such as a 2'-OMe nucleotide. The modified siRNA can then be contacted with mammalian responder cells as described above to confirm that its immunostimulatory properties have been reduced or suppressed.

[0237] Suitable in vitro assays for detecting an immune response include the double monoclonal antibody sandwich immunoassay method of 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 of Gordon et al. (U.S. Patent No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., J. Biol. Chem., 255:4980 - 4983 (1980)); the enzyme - linked immunosorbent assay (ELISA) described, for example, by Raines et al., J. Biol. Chem., 257:5154 - 5160 (1982); immunocytochemistry methods including the use of fluorescent dyes (Brooks et al., Clin. Exp. Immunol., 39:477 (1980)); and neutralization of activity (Bowen - Pope et al., Proc. Natl. Acad. Sci. USA, 81:2396 - 2400 (1984)), but are not limited thereto. In addition to the aforementioned immunoassays, a number of other immunoassays are available, 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 hereby incorporated by reference in their entirety for all purposes.

[0238] 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, an assay that can be performed as follows: (1) siRNA can be administered to 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, N.J.), human IL-6 and TNF-α (eBioscience; San Diego, Calif.), and mouse IL-6, TNF-α, and IFN-γ (BD Biosciences; San Diego, Calif.)).

[0239] Monoclonal antibodies that specifically bind to cytokines and growth factors are commercially available from a plurality of 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 production of monoclonal antibodies has been previously described and can be performed 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 with a composition that facilitates detection (e.g., any composition detectable by spectroscopic, photochemical, biochemical, electrical, optical, or chemical means).

[0240] Production of siRNA Molecules siRNA can be provided in several forms, for example, as one or more isolated small interfering RNA (siRNA) duplexes, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a transcription cassette in a DNA plasmid. The siRNA sequence 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., have blunt ends).

[0241] A population of RNAs can be used to obtain long precursor RNAs, or siRNAs can be generated using long precursor RNAs having substantial or complete identity to a selected target sequence. RNAs can be isolated, synthesized, and / or cloned from cells or tissues according to methods well known to those of skill in the art. The RNAs can be a mixed population (such as obtained from cells or tissues, transcribed from cDNA, subtracted, selected, etc.), or can represent a single target sequence. RNAs can be natural (e.g., isolated from a tissue or cell sample), synthesized in vitro (e.g., using T7 or SP6 polymerase and a PCR product or cloned cDNA), or chemically synthesized.

[0242] For synthetic RNAs, to form long dsRNAs, the complement is also transcribed in vitro and hybridized to form dsRNA. When using a natural RNA population, the RNA complement is also provided, for example, by transcribing the cDNA corresponding to the RNA population or by using RNA polymerase (to form dsRNA for digestion, e.g., by E. coli RNAseIII or Dicer). The precursor RNAs are then hybridized to form double-stranded RNA for digestion. The dsRNA can be administered directly to the subject or digested in vitro prior to administration.

[0243] Methods for RNA isolation, RNA synthesis, nucleic acid hybridization, cDNA library construction and screening, and performance of PCR are well known in the art, similar to the PCR method (see U.S. Patent Nos. 4,683,195 and 4,683,202, PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). For example, see Gubler and Hoffman, Gene, 25:263-269 (1983), Sambrook et al., supra, Ausubel et al., supra. Expression libraries are also well known to those skilled in the art. Additional basic documents disclosing general methods of use 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 hereby incorporated by reference in their entirety for all purposes.

[0244] Preferably, the siRNA is chemically synthesized. The oligonucleotides containing the siRNA molecules of the present invention can be synthesized using any of a variety of techniques known in the art, such as those described in Usman et al., J. Am. Chem. Soc., 109:7845 (1987), Scaringe et al., Nucl. Acids Res., 18:5433 (1990), Wincott et al., Nucl. Acids Res., 23:2677-2684 (1995), and Wincott et al., Methods Mol. Bio., 74:59 (1997). The synthesis of oligonucleotides uses common nucleic acid protecting groups and coupling groups such as dimethoxytrityl at the 5' end and phosphoramidite at the 3' end. As a non-limiting example, small-scale synthesis can be carried out on an Applied Biosystems synthesizer using a 0.2 μmol scale protocol. Alternatively, synthesis on a 0.2 μmol scale can be carried out on 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 of skill in the art.

[0245] siRNA molecules can also be synthesized via tandem synthesis techniques, where both strands are synthesized as a single continuous oligonucleotide fragment or strand separated by a cleavable linker, which are then cleaved to yield separate fragments or strands that hybridize to form the siRNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. Tandem synthesis of siRNA can be readily adapted to both multiwell / multiplate synthesis platforms and large-scale synthesis platforms using batch reactors, synthesis columns, etc. Alternatively, siRNA molecules 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 ligated together by hybridization or ligation after synthesis and / or deprotection. In certain other cases, siRNA molecules can be synthesized as a single continuous oligonucleotide fragment, where self-complementary sense and antisense regions hybridize to form an siRNA duplex with a hairpin secondary structure.

[0246] Modification of the siRNA sequence In certain embodiments, the siRNA molecule comprises a duplex having two strands and at least one modified nucleotide in the duplex region, wherein each strand is from about 15 to about 60 nucleotides in length. Advantageously, the modified siRNA is less immunostimulatory 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 immunostimulatory properties of the siRNA and the retention of RNAi activity. As a non-limiting example, an siRNA molecule targeting a gene of interest can be minimally modified at selected uridine and / or guanosine nucleotides within the siRNA duplex (e.g., less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% modified) while retaining its ability to silence target gene expression and eliminating the immune response generated by the siRNA.

[0247] 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. For example, 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) nucleotides), 2'-O-(2-methoxyethyl) (MOE) nucleotides, 2'-methyl-thio-ethyl nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'-deoxy-2'-chloro (2'Cl) nucleotides, and 2'-azido nucleotides. In certain cases, the siRNA molecules described herein contain one or more G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog, the modification of which confers the ability to hydrogen bond to both the Watson-Crick and Hoogsteen faces of complementary guanine nucleotides within a duplex (see, for example, Lin et al., J. Am. Chem. Soc., 120:8531-8532 (1998)). Furthermore, nucleotide base analogs, such as C-phenyl, C-naphthyl, and other aromatic derivatives, inosine, azole carboxamide, and nucleotides having 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.

[0248] In certain embodiments, the siRNA molecule may further comprise one or more chemical modifications, such as terminal cap moieties, phosphate backbone modifications, etc. Examples of terminal cap moieties include inverted deoxyabasic residues, glyceryl modifications, 4’,5’-methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4’-thio nucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3’,4’-seco nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3’-3’-inverted nucleotide moieties, 3’-3’-inverted abasic moieties, 3’-2’-inverted nucleotide moieties, 3’-2’-inverted abasic moieties, 5’-5’-inverted nucleotide moieties, 5’-5’-inverted abasic moieties, 3’-5’-inverted deoxyabasic 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’-phosphoramidates, 5’-phosphoramidates, hexyl phosphates, aminohexyl phosphates, 3’-phosphates, 5’-amino, 3’-phosphorothioates, 5’-phosphorothioates, phosphorodithioates, and bridged or unbridged methylphosphonates or 5’-mercapto moieties, but are not limited thereto (see, e.g., 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 linkages) include phosphorothioates, phosphorodithioates, methylphosphonates, phosphotriesters, morpholinos, amidates, carbamates, carboxymethyl, acetamidates, 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' termini of the sense strand, antisense strand, or both strands of the siRNA. The disclosures of these references are hereby incorporated by reference in their entirety for all purposes.

[0249] In some embodiments, the sense strand and / or antisense strand of the siRNA molecule can further include a 3' terminal overhang having from 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 siRNA molecules are described, for example, in UK Patent No. GB2,397,818B and U.S. Patent Application Publication Nos. 20040192626, 20050282188, and 20070135372, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

[0250] 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 strand in place of one or more nucleotide units, including sugar and / or phosphate substitutions, and that allows the remaining bases to exhibit their activity. This group or compound does not contain commonly recognized nucleotide bases such as adenosine, guanine, cytosine, uracil, or thymine, and is thus abasic in that it lacks a base at the 1' position.

[0251] In other embodiments, chemical modification of the siRNA includes attaching a conjugate to the siRNA molecule. The conjugate can be attached to the 5' and / or 3' end of the sense and / or antisense strand of the siRNA via a covalent bond, such as a biodegradable linker. The conjugate can also be attached to the siRNA via, for example, a carbamate group or other linking group (see, e.g., U.S. Patent Application Publication Nos. 20050074771, 20050043219, and 20050158727). In certain cases, the conjugate is a molecule that facilitates delivery of the siRNA into cells. Examples of conjugate molecules suitable for attachment 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 derivatives thereof), saccharides (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, e.g., U.S. Patent Application Publication Nos. 20030130186, 20040110296, and 20040249178, U.S. Patent No. 6,753,423). Other examples include lipophilic moieties, vitamins, polymers, peptides, proteins, nucleic acids, small molecules, oligosaccharides, carbohydrate clusters, intercalators, minor groove binders, cleaving agents, and cross-linking agent conjugate molecules described in U.S. Patent Application Publication Nos. 20050119470 and 20050107325. Still other examples include 2'-O-alkylamines, 2'-β-alkoxyalkylamines, polyamines, C5-cationic modified pyrimidines, cationic peptides, guanidium groups, amidininium groups, and cationic amino acid conjugate molecules described in U.S. Patent Application Publication No. 20050153337. Additional examples include conjugate molecules comprising a hydrophobic group, a membrane active compound, a cell permeable compound, a cell targeting signal, an interaction modifier, and a steric stabilizer as described in U.S. Patent Application Publication No. 20040167090. Further examples include conjugate molecules as 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 improved pharmacokinetic profiles, bioavailability, and / or stability of the siRNA while retaining RNAi activity. Thus, one of ordinary skill in the art can use any of a variety of well-known in vitro cell cultures or in vivo animal models to screen siRNA molecules conjugated with various conjugates and identify those having improved properties and full RNAi activity. The disclosures of the foregoing patent documents are hereby incorporated by reference in their entirety for all purposes.

[0252] Target gene In certain embodiments, the nucleic acid component (e.g., siRNA) of the nucleic acid-lipid particles described herein can be used to down-regulate or silence the translation (i.e., expression) of a gene of interest. Genes of interest include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., liver diseases and disorders), genes associated with tumor formation and cell transformation (e.g., cancer), angiogenesis genes, immunomodulatory genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders. In certain embodiments, the gene of interest is expressed in hepatocytes.

[0253] Genes related to virus infection and survival include genes that bind, enter, and replicate within cells and are expressed by the virus. Particular targets are virus sequences associated with chronic viral diseases. Particularly targeted virus 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, Furin virus, Machupo virus, Guanarito virus, 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)), 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 Virol., 83:2635-2662 (2002)), hepatitis viruses (see, for example, 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 ed., Lippincott-Raven, Philadelphia (2001)), human immunodeficiency virus (HIV) (Banerjea et al., Mol. Ther., 8:62 (2003), Song et al., J. Virol., including the sequences of 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)), as well as those of ,77:7174(2003), Stephenson, JAMA, 289:1494(2003), Qin et al., Proc. Natl. Acad. Sci. USA, 100:183(2003)).

[0254] 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 genomic sequences of Ebola virus are 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 numbers 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. Additional Ebola virus sequences are described, for example, in Genbank accession numbers L11365 and X61274. The complete genomic sequences of Marburg virus are described, for example, in Genbank accession numbers NC_001608, AY430365, AY430366, and AY358025. The sequence of Marburg virus GP is described, for example, in Genbank accession numbers AF005734, AF005733, and AF005732.The sequences of Marburg virus VP35 are described, for example, in Genbank accession numbers AF005731 and AF005730. Additional 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 No. 20070135370, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0255] Exemplary influenza virus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences encoding nucleoprotein (NP), matrix proteins (M1 and M2), non-structural proteins (NS1 and NS2), RNA polymerase (PA, PB1, PB2), neuraminidase (NA), and hemagglutinin (HA). The NP sequence of influenza A is described, for example, in Genbank accession numbers NC_004522, AY818138, AB166863, AB188817, AB189046, AB189054, AB189062, AY646169, AY646177, AY651486, AY651493, AY651494, AY651495, AY651496, AY651497, AY651498, AY651499, AY651500, AY651501, AY651502, AY651503, AY651504, AY651505, AY651506, AY651507, AY651509, AY651528, AY770996, AY790308, AY818138, and AY818140. The PA sequence of influenza A is described, for example, in Genbank accession numbers AY818132, AY790280, AY646171, AY818132, AY818133, AY646179, AY818134, AY551934, AY651613, AY651610, AY651620, AY651617, AY651600, AY651611, AY651606, AY651618, AY651608, AY651607, AY651605, AY651609, AY651615, AY651616, AY651640, AY651614, AY651612, AY651621, AY651619, AY770995, and AY724786. Non-limiting examples of siRNA molecules targeting influenza virus nucleic acid sequences include those described in U.S. Patent Application Publication No. 20070218122, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0256] Exemplary nucleic acid sequences of hepatitis viruses that can be silenced include nucleic acid sequences involved in transcription and translation (e.g., En1, En2, X, P), as well as nucleic acid sequences encoding 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), but are not limited thereto (see, e.g., FIELDS VIROLOGY, supra). Exemplary nucleic acid sequences of hepatitis C virus (HCV) that can be silenced include 5' untranslated region (5'UTR), 3' untranslated region (3'UTR), polyprotein translation initiation codon region, internal ribosome entry site (IRES) sequence, and / or nucleic acid sequences encoding core protein, E1 protein, E2 protein, p7 protein, NS2 protein, NS3 protease / helicase, NS4A protein, NS4B protein, NS5A protein, and / or NS5B RNA-dependent RNA polymerase, but are not limited thereto. The genomic sequence of HCV 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 viral nucleic acid sequence of hepatitis A is described, for example, in Genbank accession number NC_001489, the nucleic acid sequence of hepatitis B virus is described, for example, in Genbank accession number NC_003977, the nucleic acid sequence of hepatitis D virus is described, for example, in Genbank accession number NC_001653, the nucleic acid sequence of hepatitis E virus is described, for example, in Genbank accession number NC_001434, and the nucleic acid sequence of hepatitis G virus is described, for example, in Genbank accession number NC_001710.Silencing of sequences encoding genes related to viral infection and survival can be advantageously used in combination with the administration of conventional agents used to treat the viral condition. Non-limiting examples of siRNA molecules targeting hepatitis virus nucleic acid sequences include those described in U.S. Patent Application Publication Nos. 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 hereby incorporated by reference in their entirety for all purposes.

[0257] Genes associated with metabolic diseases and disorders (e.g., disorders where the liver is the target, as well as liver diseases and disorders) include, for example, genes that are 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 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) (see, for example, 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 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 where the liver is the target, as well as liver diseases and disorders) include genes that are expressed in the liver itself as well as genes that are expressed in other organs and tissues.Silencing of sequences encoding genes associated with metabolic diseases and disorders can be advantageously used in combination with the administration of conventional agents 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 No. 20060134189, the disclosure of which is hereby incorporated 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 No. 61 / 147,235, filed January 26, 2009, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0258] Examples of gene sequences associated with tumor formation and cell transformation (e.g., cancer or other neoplasia) 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); COP9 signalosome subunits such as CSN1, CSN2, CSN3, CSN4, CSN5 (JAB1; Genbank accession number NM_006837), 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, HDAC9, and the like. 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 May 29, 2007, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. Non-limiting examples of siRNA molecules targeting the PLK-1 gene include those described in U.S. Patent Application Publication Nos. 20050107316 and 20070265438, and U.S. Patent Application No. 12 / 343,342, filed December 23, 2008, the disclosures of which are hereby incorporated 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 No. 61 / 045,251, filed April 15, 2008, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0259] Further examples of gene sequences associated with tumor formation and cell transformation include translocation sequences, such as the MLL fusion gene, 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, such as the multidrug resistance gene (Nieth et al., FEBS Lett., 545:144 (2003), Wu et al, Cancer Res. 63:1515 (2003)), cyclin (Li et al., Cancer Res., 63:3593 (2003), Zou et al., Genes Dev., 16:2923 (2002)), beta-catenin (Verma et al., Clin Cancer Res., 9:1291 (2003)), the telomerase gene (Kosciolek et al., Mol Cancer Ther., 2:209 (2003)), c-MYC, N-MYC, BCL-2, growth factor receptors (e.g., EGFR / ErbB1 (Genbank accession numbers NM_005228, NM_201282, NM_201283, and NM_201284, see also Nagy et al. Exp. Cell Res., 285:39-49 (2003)), ErbB2 / HER-2 (Genbank accession 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 mutant sequences such as RAS (reviewed 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 May 29, 2007, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0260] Silencing of 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 involved in tumor migration are also target sequences of interest, such as integrins, selectins, and metalloproteinases. The foregoing examples are not exclusive. Those skilled in the art will understand that any whole or partial gene sequence that promotes or facilitates tumorigenesis or cell transformation, tumor growth, or tumor migration may be included as a template sequence.

[0261] Angiogenic genes can promote the formation of new blood vessels. Particular targets 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 hereby incorporated by reference in their entirety for all purposes.

[0262] Anti-angiogenic genes can inhibit neovascularization. These genes are particularly useful in the treatment of cancers where angiogenesis plays a role in the pathological development of the disease. Examples of anti-angiogenic genes include, but are not limited to, endostatin (see, e.g., U.S. Patent No. 6,174,861), angiostatin (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 of which are hereby incorporated by reference in their entirety for all purposes.

[0263] Immune regulatory genes are genes that regulate one or more immune responses. Examples of immune regulatory genes include cytokines such as 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 TNF, but are not limited thereto. The Fas and Fas ligand genes are also immune regulatory target sequences of interest (Song et al., Nat. Med., 9:347 (2003)). Genes encoding secondary signaling molecules in hematopoietic and lymphoid cells, such as Tec family kinases such as Bruton's tyrosine kinase (Btk) (Heinonen et al., FEBS Lett., 527:274 (2002)), are also included in the present invention.

[0264] Cell receptor ligands include ligands that can bind to cell surface receptors (e.g., insulin receptor, EPO receptor, G protein-coupled receptor, receptor having tyrosine kinase activity, cytokine receptor, growth factor receptor, etc.) and regulate (e.g., inhibit, activate, etc.) physiological pathways (e.g., regulation of glucose levels, hematopoiesis, mitogenesis, etc.) in which the receptor is involved. Examples of cell receptor ligands include cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G protein-coupled receptor ligands, etc., but are not limited thereto. Templates encoding the elongation of trinucleotide repeats (e.g., CAG repeats) are used for silencing pathogenic sequences in neurodegenerative disorders caused by the elongation of trinucleotide repeats such as spinal muscular atrophy and Huntington's disease (Caplen et al., Hum. Mol. Genet., 11:175 (2002)).

[0265] Other specific target genes that can be targeted by nucleic acids (e.g., by siRNA) to downregulate or silence gene expression include alpha-actin-2 of aorta smooth muscle (ACTA2), alcohol dehydrogenase 1A (ADH1A), alcohol dehydrogenase 4 (ADH4), alcohol dehydrogenase 6 (ADH6), afamin (AFM), angiotensinogen (AGT), serine-pyruvate aminotransferase (AGXT), alpha-2-HS-glycoprotein (AHSG), aldo-keto reductase family 1 member C4 (AKR1C4), serum albumin (ALB), alpha-1-microglobulin / bikunin precursor (AMBP), angiopoietin-related protein 3 (ANGPTL3), serum amyloid P component (APCS), apolipoprotein A-II (APOA2), apolipoprotein B-100 (APOB), apolipoprotein C-III (APOC3), apolipoprotein C-IV (APOC4), apolipoprotein F (APOF), beta-2-glycoprotein 1 (APOH), aquaporin-9 (AQP9), bile acid-CoA:amino acid N-acyltransferase (BAAT), C4b-binding protein beta chain (C4BPB), putative protein of unknown nature encoded by LINC01554 (C5orf27), 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 (C8G), 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), C-X-C 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 1 (FGL1), flavin-containing monooxygenase 3 (FMO3), flavin-containing monooxygenase 5 (FMO5), 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), hydroxyacid oxidase (glycolic acid oxidase) 1 (HAO1), HGF activator (HGFAC), haptoglobin-related protein; haptoglobin (HPR), hemopexin (HPX), histidine-rich glycoprotein (HRG), hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1), hydroxysteroid (17-beta) dehydrogenase 13 (HSD17B13), inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1), inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), prekallikrein (KLKB1), lactate dehydrogenase A (LDHA), liver-expressed antimicrobial peptide 2 (LEAP2), leukocyte cell-derived chemotaxin 2 (LECT2), lipoprotein(a) (LPA), mannan-binding lectin serine peptidase 2 (MASP2), S-adenosylmethionine synthase isoform 1 type (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)Composed of serum amyloid A4 (SAA4), serine dehydratase (SDS), serpin family A member 1 (SERPINA1), serpin A11 (SERPINA11), calistatin (SERPINA4), corticosteroid-binding globulin (SERPINA6), antithrombin III (SERPINC1), heparin cofactor 2 (SERPIND1), serpin 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 facilitative glucose transporter member 2 (SLC2A2), sodium-coupled neutral amino acid transporter 4 (SLC38A4), solute carrier organic anion transporter family member 1B1 (SLCO1B1), sphingomyelin phosphodiesterase 1 (SMPD1), bile acid 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), but not limited to these.

[0266] In addition to its 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 in research and development applications, as well as in diagnostic, preventive, prognostic, clinical, and other health management applications. As a non-limiting example, the specific nucleic acids (e.g., siRNA) can be used in target validation assays aimed at testing whether a gene of interest may be a therapeutic target. The specific nucleic acids (e.g., siRNA) can also be used in target identification assays aimed at finding genes as potential therapeutic targets.

[0267] CRISPR Targeted genome editing has evolved from a niche technology to a method used by many biological researchers. This evolution has been greatly facilitated by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR) technology (see, e.g., Sander et al., Nature Biotechnology, 32(4), 347-355 including Supplementary Information (2014), International Publication Nos. WO2016 / 197132 and WO2016 / 197133). Accordingly, provided herein are improvements (e.g., lipid nanoparticles and formulations thereof) that can be used in combination with CRISPR technology to treat diseases such as HBV. With respect to targets for using CRISPR, the guide RNA (gRNA) utilized in CRISPR technology can be designed to target a specifically identified sequence, e.g., a target gene (e.g., a target gene of the HBV genome). Examples of such target sequences are shown in International Publication No. WO2016 / 197132. Further, International Publication No. WO2013 / 151665 (see, e.g., Table 6, which is specifically incorporated by reference in its entirety, including Table 6 and the accompanying sequence listing) describes approximately 35,000 mRNA sequences that are claimed in relation to mRNA expression constructs. Certain embodiments of the invention utilize CRISPR technology to target the expression of any of these sequences. Certain embodiments of the invention can also utilize CRISPR technology to target the expression of the target genes discussed herein.

[0268] aiRNA Similar to siRNA, asymmetric interfering RNA (aiRNA) can recruit the RNA-induced silencing complex (RISC) and effectuate efficient silencing of various genes in mammalian cells by mediating sequence-specific cleavage of the 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, an aiRNA molecule comprises a short RNA duplex having a sense strand and an antisense strand, the duplex containing overhangs at the 3'- and 5'-ends of the antisense strand. aiRNA is generally asymmetric, as 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. By way of non-limiting example, aiRNA sequences can be selected and generated using the aforementioned methods for selecting siRNA sequences.

[0269] In another embodiment, aiRNA duplexes of various 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 target a desired mRNA, including overhangs at the 3'- and 5'-ends of the antisense strand. In certain cases, the sense strand of the aiRNA molecule is about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain other cases, the antisense strand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotides in length, more typically about 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length.

[0270] 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 aspects, the aiRNA molecules described herein may include one or more modified nucleotides, for example, in the double-stranded (duplex) region and / or in the antisense overhang. By way of non-limiting example, the aiRNA sequence may include one or more of the modified nucleotides described above for the siRNA sequence. In preferred embodiments, the aiRNA molecule includes 2'-OMe nucleotides such as, for example, 2'-OMe-guanosine nucleotides, 2'-OMe-uridine nucleotides, or mixtures thereof.

[0271] In certain embodiments, the aiRNA molecule may include an antisense strand corresponding to the antisense strand of the 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 associated with viral infection and survival, genes associated with metabolic diseases and disorders, genes associated with tumor formation and cell transformation, angiogenesis genes, immune regulatory genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.

[0272] miRNA Generally, microRNA (miRNA) is a single-stranded RNA molecule about 21 to 23 nucleotides in length that controls gene expression. miRNA is encoded by a gene, and miRNA is transcribed from the DNA of that gene, but miRNA is not translated into protein (non-coding RNA). Instead, each primary transcript (pri-miRNA) is processed into a short stem-loop structure called pre-miRNA and ultimately into a functional mature miRNA. Mature miRNA molecules are either partially or fully complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression. The 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.

[0273] Genes encoding miRNA are much longer than the processed mature miRNA molecules. miRNA is first transcribed as a primary transcript or pri-miRNA with a cap and polyA tail and is processed in the cell nucleus into a short stem-loop structure of about 70 nucleotides known as pre-miRNA. This processing is carried out in animals by a protein complex known as the microprocessor complex, consisting of the nuclease Drosha and the double-stranded RNA-binding protein Pasha (Denli et al., Nature, 432:231-235 (2004)). These pre-miRNAs are then processed in the cytoplasm into mature miRNAs by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC) (Bernstein et al., Nature, 409:363-366 (2001). Either the sense or antisense strand of the DNA can function as a template for generating miRNA.

[0274] When a dicer cuts a pre-miRNA stem loop, two complementary short RNA molecules are formed, but only one of them is incorporated into the RISC complex. This strand, known as the guide strand, is selected by the argonaute 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, miRNAs base pair with their complementary mRNA molecules and induce degradation and / or translational silencing of the target mRNA.

[0275] Mammalian miRNA molecules are usually complementary to sites within the 3' UTR of the target mRNA sequence. In certain cases, annealing of the miRNA to the target mRNA inhibits protein translation by blocking the protein translation machinery. In certain other cases, annealing of the miRNA to the 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 association with a complement of proteins collectively referred to as miRNP.

[0276] In certain embodiments, the miRNA molecules described herein are about 15 to 100, 15 to 90, 15 to 80, 15 to 75, 15 to 70, 15 to 60, 15 to 50, or 15 to 40 nucleotides in length, more typically about 15 to 30, 15 to 25 or 19 to 25 nucleotides in length, preferably about 20 to 24, 21 to 22, or 21 to 23 nucleotides in length. In certain other embodiments, the miRNA molecule may contain one or more modified nucleotides. By way of non-limiting example, the miRNA sequence may contain one or more of the modified nucleotides described above for siRNA sequences. In preferred embodiments, the miRNA molecule contains 2′OMe nucleotides such as, for example, 2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, or mixtures thereof.

[0277] In some embodiments, the miRNA molecules are used to silence the expression of any of the aforementioned target genes, such as genes associated with viral infection and survival, genes associated with metabolic diseases and disorders, genes associated with tumor formation and cell transformation, angiogenesis genes, immunomodulatory genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.

[0278] In other embodiments, one or more agents that block the activity of the miRNA targeting the mRNA of interest are administered using the lipid particles (e.g., nucleic acid-lipid particles) of the invention. Examples of blockers include, but are not limited to, steric blocking oligonucleotides, locked nucleic acid oligonucleotides, and morpholino oligonucleotides. Such blockers may bind directly to the miRNA or to the miRNA binding site on the target mRNA.

[0279] antisense oligonucleotide In one embodiment, the nucleic acid is an antisense oligonucleotide targeting a target gene or sequence of interest. The term "antisense oligonucleotide" or "antisense" includes 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 blocks translation of the complementary RNA strand by binding to that RNA. Antisense DNA oligonucleotides can be used to target specific complementary (coding or non-coding) RNAs. When binding occurs, this DNA / RNA hybrid can be degraded by the enzyme RNase H. In certain embodiments, the antisense oligonucleotide comprises from about 10 to about 60 nucleotides, more preferably from about 15 to about 30 nucleotides. The term also encompasses antisense oligonucleotides that may not be strictly complementary to the desired target gene. Thus, the present invention is available when non-target-specific activity is seen with the antisense or when an antisense sequence containing one or more mismatches with the target sequence is most preferred for a particular use.

[0280] 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 a target gene. The effectiveness of antisense oligonucleotides for inhibiting protein synthesis is well established. For example, the synthesis of polygalactauronase and the muscarinic type 2 acetylcholine receptor are inhibited by antisense oligonucleotides directed against their respective mRNA sequences (see U.S. Patent Nos. 5,739,119 and 5,759,829). Further, examples of antisense inhibition are shown with nuclear protein cyclin, the multidrug resistance gene (MDR1), ICAM-1, E-selectin, STK-1, striatal GABAA receptor, and human EGF (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 see U.S. Patent Nos. 5,801,154, 5,789,573, 5,718,709, and 5,610,288). Further, antisense constructs have been described that can be used to inhibit and treat various abnormal cell proliferations, such as cancer (see U.S. Patent Nos. 5,747,470, 5,591,317, and 5,783,683). The disclosures of these references are hereby incorporated by reference in their entirety for all purposes.

[0281] Methods for generating antisense oligonucleotides are known in the art and can be readily adapted to generate antisense oligonucleotides that target any polynucleotide sequence. The selection of an antisense oligonucleotide sequence specific for a given target sequence involves analysis of the selected target sequence, as well as secondary structure, T m, 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 prevent specific binding to target mRNA within a host cell. Highly preferred target regions of mRNA include regions in or near the AUG translation initiation codon and sequences substantially complementary to the 5' region of the mRNA. Consideration of these secondary structure analyses and target site selections can be performed, for example, using v.4 of OLIGO Primer Analysis Software (Molecular Biology Insights) and / or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).

[0282] Ribozyme According to another embodiment of the present invention, the nucleic acid-lipid particles are associated with a ribozyme. A ribozyme is an RNA-protein complex 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 phosphoester transfer reactions with a high degree of specificity and often cleave only one of several phosphoesters within an oligonucleotide substrate (see Cech et al., Cell, 27:487-96 (1981), Michel et al., J. Mol. Biol., 216:585-610 (1990), Reinhold-Hurek et al., Nature, 357:173-6 (1992)). This specificity results from the requirement that the substrate binds via base pair interactions specific to the internal guide sequence ("IGS") of the ribozyme prior to the chemical reaction.

[0283] Currently, at least six basic types of natural enzymatic RNA molecules are known. Each can catalyze in trans the hydrolysis of RNA phosphodiester bonds (and thus can cleave other RNA molecules). Generally, an enzymatic nucleic acid acts first by binding to a target RNA. Such binding occurs through the 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, an enzymatic nucleic acid first recognizes the target RNA, then binds to it via complementary base pairing, and when 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 binds to and cleaves its RNA target, the enzymatic nucleic acid can be released from that RNA, search for another target, and repeatedly bind to and cleave new targets.

[0284] The enzymatic nucleic acid molecules can be formed, for example, of a hammerhead, hairpin, hepatitis delta virus, group I intron or RNaseP RNA (associated with an RNA guide sequence) or Neurospora VS RNA motif. Specific examples of the hammerhead motif are described, for example, in Rossi et al., Nucleic Acids Res., 20:4559-65 (1992). Examples of the hairpin motif 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 the hepatitis delta virus motif are described, for example, in Perrotta et al., Biochemistry, 31:11843-52 (1992). Examples of the RNaseP motif are described, for example, in Guerrier-Takada et al., Cell, 35:849-57 (1983). Examples of the Neurospora VS RNA ribozyme motif 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), 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 specific substrate binding sites that are complementary to one or more of the DNA or RNA regions of the target gene, and that they have a nucleotide sequence within or around the substrate binding site that confers RNA cleavage activity on the molecule. Thus, ribozyme constructs are not necessarily limited to the specific motifs mentioned herein. The disclosures of these references are hereby incorporated by reference in their entirety for all purposes.

[0285] Methods for generating ribozymes that target any polynucleotide sequence are known in the art. Ribozymes can be designed as described, for example, in PCT Publications WO93 / 23569 and WO94 / 02595 and synthesized for testing in vitro and / or in vivo as described therein. The disclosures of these PCT Publications are hereby incorporated by reference in their entirety for all purposes.

[0286] Ribozyme activity can be optimized by chemically synthesizing ribozymes having modifications that change the length of the ribozyme binding arms, or that prevent 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 moiety of an enzymatic RNA molecule, and the disclosures of which are hereby incorporated by reference in their entirety for all purposes), modifications that enhance their effectiveness intracellularly, and removal of stem II bases for shortening RNA synthesis time and reducing chemical requirements.

[0287] Immunostimulatory oligonucleotide The nucleic acid associated with the lipid particles of the invention can be immunostimulatory, including immunostimulatory oligonucleotides (ISS; single-stranded or double-stranded) that can induce an immune response when administered to a subject, which can be a mammal such as a human. ISS includes, for example, specific palindromic structures that result in a hairpin secondary structure (see Yamamoto et al., J. Immunol., 148:4072-6 (1992)), or CpG motifs, and other known features of ISS (e.g., multiple G domains; see PCT Publication WO96 / 11266, the disclosure of which is hereby incorporated by reference in its entirety for all purposes).

[0288] Immunostimulatory nucleic acids are considered non-sequence-specific when they do not need to specifically bind to a target sequence to reduce its expression in order to induce an immune response. Thus, certain immunostimulatory nucleic acids may contain sequences corresponding to regions of natural genes or mRNAs, but they can still be considered non-sequence-specific immunostimulatory nucleic acids.

[0289] In one embodiment, the immunostimulatory nucleic acid or oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may or may not be methylated. 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 the cytosines in this CpG dinucleotide is methylated. In a further embodiment, each cytosine in the CpG dinucleotides present within the sequence is methylated. In another embodiment, the nucleic acid comprises a plurality of CpG dinucleotides, and at least one of the CpG dinucleotides comprises methylated cytosine. Examples of immunostimulatory oligonucleotides suitable for use in the compositions and methods of the present invention are described in PCT Application No. PCT / US08 / 88676, filed December 31, 2008, PCT Publication Nos. WO02 / 069369 and WO01 / 15726, U.S. Patent No. 6,406,705, and Raney et al., J. Pharm. Exper. Ther., 298:1185-92 (2001), the disclosures of which are hereby incorporated by reference in their entireties for all purposes. In certain embodiments, the oligonucleotides used in the compositions and methods of the present invention have a phosphodiester ("PO") backbone or a phosphorothioate ("PS") backbone, and / or at least one methylated cytosine residue within the CpG motif.

[0290] mRNA In certain embodiments, the nucleic acid is one or more mRNA molecules (e.g., a cocktail of mRNA molecules).

[0291] Modifications to mRNA The mRNA used in the practice of the present invention can include one, two, or more than two nucleoside modifications. In some embodiments, the modified mRNA exhibits reduced degradation in the cells into which the mRNA is introduced as compared to the corresponding unmodified mRNA.

[0292] In some embodiments, modified nucleosides include pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

[0293] In some embodiments, the modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0294] 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, N6-(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.

[0295] 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)-pseudouridine. The α-thio-substituted phosphate moiety is provided to confer stability to the RNA polymer via non-natural phosphorothioate backbone linkages. Phosphorothioate RNA has increased nuclease resistance and thus a longer half-life in the cellular environment. Phosphorothioate-linked nucleic acids are also expected to reduce the innate immune response through relatively weak binding / activation of the cell's innate immune molecules.

[0296] In certain embodiments, for example, when precise timing of protein production is desired, it may be desirable to degrade the modified nucleic acid introduced into the cell intracellularly. Accordingly, the present invention provides modified nucleic acids containing a degradation domain that can act in an intracellularly directed manner.

[0297] In other embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-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.

[0298] Any component of the modified nucleic acid In further embodiments, the modified nucleic acid can include other optional components that can be beneficial in some embodiments. These optional components include, but are not limited to, untranslated regions, Kozak sequences, intron nucleotide sequences, internal ribosome entry sites (IRES), caps, and polyA tails. For example, a 5' untranslated region (UTR) and / or a 3' UTR can be provided, and either or both of these can independently include one or more different nucleoside modifications. In such embodiments, the nucleoside modifications can also be present in the translatable region. Nucleic acids containing Kozak sequences are also provided.

[0299] Furthermore, nucleic acids containing one or more intron nucleotide sequences that can be excised from the nucleic acid are provided.

[0300] Untranslated region (UTR) The untranslated region (UTR) of a gene is transcribed but not translated. The 5' UTR starts at the transcription start site and continues to the start codon, but does not include the start codon. On the other hand, the 3' UTR starts immediately after the stop codon and continues to the transcription termination signal. There is increasing evidence for the regulatory role that UTRs play with respect to the stability and translation of nucleic acid molecules. To increase the stability of the molecule, the regulatory function of the UTR can be incorporated into the mRNA used in the present invention. It is also possible to incorporate specific functions to ensure the downregulation of transcripts when misinduced in unwanted organ sites.

[0301] 5' capping The 5' cap structure of mRNA is involved in nuclear export, increases the stability of mRNA, binds to the mRNA cap-binding protein (CBP), which is involved in the stability and translational ability of intracellular mRNA through the association of CBP with poly(A) binding protein to form mature circular mRNA species. The cap also aids in the removal of the 5' proximal intron during mRNA splicing.

[0302] Endogenous mRNA molecules can have a 5’-terminal capping such that a 5’-ppp-5’-triphosphate bond occurs between the terminal guanosine cap residue and the transcribed sense nucleotide at the 5’-end of the mRNA molecule. This 5’-guanylic acid cap can then be methylated to yield an N7-methyl-guanylic acid residue. The ribose sugar of the 5’-end of the mRNA and / or the transcribed nucleotides immediately preceding the end can also optionally be 2’-O-methylated. Hydrolysis and cleavage of the guanylic acid cap structure for 5’-cap removal can target nucleic acid molecules such as mRNA molecules for degradation.

[0303] IRES sequence mRNAs containing an internal ribosome entry site (IRES) are also useful in the practice of the present invention. The IRES may function as the sole ribosome binding site or as one of multiple ribosome binding sites of the mRNA. An mRNA containing multiple functional ribosome binding sites can encode several peptides or polypeptides that are translated independently by ribosomes (“polycistronic mRNA”). When an mRNA is provided with an IRES, optionally, a second translatable region is also provided. Examples of IRES sequences that can be used according to the present invention include, but are not limited to, those derived from picornavirus (e.g., FMDV), pest virus (CFFV), poliovirus (PV), encephalomyocarditis virus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV).

[0304] Poly-A tail During RNA processing, a long chain of adenine nucleotides (polyA tail) can be added to a polynucleotide such as an mRNA molecule to increase stability. Immediately after transcription, the 3' end of the transcript is cleaved and the 3' hydroxyl can be freed. Next, polyA polymerase adds a chain of adenine nucleotides to the RNA. In this process, called polyadenylation, a polyA tail that can be 100 - 250 residues long is added.

[0305] Generally, the length of the polyA tail exceeds 30 nucleotides. In another embodiment, the polyA tail is more than 35 nucleotides long (e.g., at least about 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 120 or more, 140 or more, 160 or more, 180 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, 1200 or more, 1300 or more, 1400 or more, 1500 or more, 1600 or more, 1700 or more, 1800 or more, 1900 or more, 2,000 or more, 2,500 or more, and 3,000 or more nucleotides).

[0306] In this context, the polyA tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the modified mRNA. The polyA tail can also be designed as part of the modified nucleic acid to which it belongs. In this context, the polyA tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90%, or more of the full length of the modified mRNA or the full length of the modified mRNA minus the polyA tail.

[0307] Production of mRNA molecules Methods for the isolation of RNA, synthesis of RNA, hybridization of nucleic acids, construction and screening of cDNA libraries, and performance of PCR are well known in the art, as are the PCR methods (see U.S. Pat. Nos. 4,683,195 and 4,683,202, PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). For example, see Gubler and Hoffman, Gene, 25:263-269 (1983), 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 general methods of use in 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 hereby incorporated by reference in their entirety for all purposes.

[0308] Encoded polypeptide Using the mRNA component of the nucleic acid-lipid particles described herein, a polypeptide of interest can be expressed. Certain diseases in humans are caused by the absence or impairment of a functional protein in the cell type in which the protein normally exists and is active. The functional protein may be completely or partially absent, for example, due to transcriptional inactivity of the encoding gene or the presence of mutations in the encoding gene that render the protein completely or partially non-functional. Examples of human diseases caused by complete or partial inactivation of a protein include X-linked severe combined immunodeficiency (X-SCID) and X-linked adrenoleukodystrophy (X-ALD). X-SCID is caused by one or more mutations in the gene encoding the common gamma chain protein, which is a component of the receptors for several interleukins involved in the development and maturation of B and T cells within the immune system. X-ALD is caused by one or more mutations in the peroxisomal membrane transporter protein gene called ABCD1. Individuals affected with X-ALD have extremely high levels of long-chain fatty acids in tissues throughout the body, which causes various symptoms that can lead to mental impairment or death.

[0309] Attempts have been made to use gene therapy to treat some diseases caused by the absence or impairment of a functional protein in the cell type in which the protein normally exists and is active. Gene therapy typically involves the introduction of a vector containing a gene encoding a functional form of the disease-causing protein into an affected individual and the expression of the functional protein to treat the disease. To date, success in gene therapy has been limited. Additionally, certain aspects of using LNPs to deliver mRNA have been described, for example, in International Publication Nos. WO2018 / 006052 and WO2015 / 011633.

[0310] Therefore, there remains a need for improvements for expressing the functional form of a protein in a human suffering from a disease caused by the complete or partial absence of a functional protein, and there is also a need for improvements in nucleic acid (e.g., mRNA) delivery via methods and compositions that can, for example, further reduce the induction of an immune response to a therapy. Certain embodiments of the present invention are useful in this context. Thus, in certain embodiments, the expression of a polypeptide ameliorates one or more symptoms of a disease or disorder. Certain compositions and methods of the present invention may be useful for treating human diseases caused by the absence or reduced levels of a functional polypeptide in the human body. In other embodiments, certain compositions and methods of the present invention may be useful for the expression of vaccine antigens (e.g., for treating cancer).

[0311] Self-amplifying RNA In certain embodiments, the nucleic acid is one or more self-amplifying RNA molecules. Self-amplifying RNA (sa-RNA) may also be referred to as self-replicating RNA, replicable RNA, replicon, or RepRNA. RepRNA, referred to as self-amplifying mRNA, when derived from a plus-strand virus, is generated from a viral genome lacking at least one structural gene, and the RepRNA can translate and replicate (and thus "self-amplify") without generating infectious progeny virus. In certain embodiments, RepRNA technology can be used to insert a gene cassette encoding a desired target antigen. For example, the alphavirus genome is divided into two open reading frames (ORFs), the first ORF encoding the protein of RNA-dependent RNA polymerase (replicase), and the second ORF encoding the structural protein. In an sa-RNA vaccine construct, the ORF encoding the viral structural protein can be replaced with any selected antigen, while the viral replicase remains an essential part of the vaccine and promotes intracellular amplification of the RNA after immunization.

[0312] Other active agents In certain embodiments, the agent that associates with the lipid particles of the present invention can include one or more therapeutic proteins, polypeptides, or small organic molecules or compounds. Non-limiting examples of such therapeutically effective agents or drugs include tumor drugs (e.g., chemotherapeutic agents, hormonal therapy agents, immunotherapy agents, radiation therapy agents, etc.), lipid-lowering agents, antiviral drugs, anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, contraceptives, antipyretics, vasodilators, anti-angiogenic drugs, cytovascular agents, signal transduction inhibitors, cardiovascular drugs such as antiarrhythmic agents, hormones, vasoconstrictors, and steroids. These agents can be administered alone with the lipid particles of the present invention or in combination (e.g., co-administered) with the lipid particles of the present invention that contain nucleic acids such as interfering RNA or mRNA.

[0313] Non-limiting examples of chemotherapeutic agents include platinum-based drugs (e.g., oxaliplatin, cisplatin, carboplatin, spiropent, iproplatin, satraplatin, etc.), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uracil mustard, thiotepa, nitrosourea, etc.), antimetabolites (e.g., 5-fluorouracil (5-FU), azathioprine, methotrexate, leucovorin, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, pemetrexed, raltitrexed, etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel (Taxol), docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan (CPT-11, Camptosar), topotecan, amsacrine, etoposide (VP16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.), tyrosine kinase inhibitors (e.g., gefitinib (Iressa (registered trademark)), sunitinib (Sutent (registered trademark), SU11248), erlotinib (Tarceva (registered trademark), OSI-1774), lapatinib (GW572016, GW2016), canertinib (CI1033), semaxinib (SU5416), brivanib (PTK787 / ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec (registered trademark), STI571), dasatinib (BMS-354825), leflunomide (SU101), vandetanib (Zactima (trademark), ZD6474), etc.), pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogs thereof, and combinations thereof are included.

[0314] Examples of conventional hormonal therapy agents 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).

[0315] Examples of conventional immunotherapy agents include, but are not limited to, immunostimulants (e.g., Bacillus Calmette-Guerin (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.), as well as radioimmunotherapy (e.g., 111 In,[[]] 90 Y, or 131 I, etc., conjugated anti-CD20 monoclonal antibodies).

[0316] Examples of conventional radiotherapy agents include those optionally conjugated to antibodies targeting tumor antigens, 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 radioactive nuclides such as Bi, but are not limited to these.

[0317] Additional tumor drugs that can be used in accordance with the present invention include, but are not limited to, Alkeran, allopurinol, altretamine, amifostine, anastrozole, araC, arsenic trioxide, bexarotene, biCNU, carmustine, CCNU, celecoxib, cladribine, cyclosporin A, cytosine arabinoside, cyclophosphamide, dexrazoxane, DTIC, estramustine, exemestane, FK506, gemtuzumab-ozogamicin, hydrea, hydroxyurea, idarubicin, interferon, letrozole, leustatin, leuprolide, litretinoin, megastrol, L-PAM, mesna, methoxsalen, mitomycin, nitrogen mustard, pamidronate, pegademase, pentostatin, porfimer sodium, prednisone, rituxan, streptozocin, STI-571, taxotere, temozolamide, VM-26, tamoxifen, tretinoin, ATRA, valrubicin, and velban. Other examples of tumor drugs that can be used in accordance with the present invention are ellipticine and ellipticine analogs or derivatives, epothilone, intracellular kinase inhibitors, and camptothecin.

[0318] Non-limiting examples of lipid-lowering agents for treating lipid diseases or disorders associated with elevated triglycerides, cholesterol, and / or glucose include statins, fibrates, ezetimibe, thiazolidinediones, niacin, beta blockers, nitroglycerin, calcium antagonists, fish oil, and mixtures thereof.

[0319] Examples of antiviral drugs include, but are not limited to, abacavir, acyclovir, aciclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripla, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fixed-dose combinations, homilersen, fosamprenavir, foscarnet, phosphonat, fusion inhibitors, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors, type III interferons (e.g., IFN-λ molecules such as IFN-λ1, IFN-λ2, and IFN-λ3), type II interferon (e.g., IFN-γ), type I interferons (e.g., IFN-α such as pegylated IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ), interferons, lamivudine, lopinavir, lobucavir, MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogs, oseltamivir, penciclovir, peramivir, pre-conalil, podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir, stubidine, synergistic enhancers, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, tridivir, tromantadine, truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, zidovudine, pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogs thereof, and mixtures thereof.

[0320] Lipid particles The lipid particles of the present invention typically comprise an active or therapeutic agent, a cationic lipid, a non-cationic lipid, and a conjugate lipid that inhibits aggregation of the particles. In some embodiments, the active or therapeutic agent is completely encapsulated within the lipid moiety of the lipid particle such that the active or therapeutic agent in the lipid particle is resistant to enzymatic degradation by, for example, nucleases or proteases in an 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.

[0321] 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 RNAs (e.g., siRNA, aiRNA, and / or miRNA) or mRNA, a cationic lipid (e.g., a cationic lipid of formula I, II, and / or III), a non-cationic lipid (e.g., cholesterol alone or a mixture of one or more phospholipids and cholesterol), and a conjugate lipid that inhibits aggregation of the particles (e.g., one or more PEG-lipid conjugates). The LNPs can comprise 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 their preparation are described, for example, in U.S. Pat. Nos. 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 No. WO96 / 40964, the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

[0322] Non-cationic lipid The non-cationic lipid used in the lipid particles (e.g., LNPs) of the present invention can be any of a variety of neutral non-charged, zwitterionic, or anionic lipids that can form stable complexes.

[0323] Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl-phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), palmitoyl oleoyl-phosphatidylglycerol (POPG), dioleoyl phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyl oleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, and mixtures thereof. Other diacyl phosphatidylcholine and diacyl phosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having a C 10 ~C 24 carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0324] Further examples of non-cationic lipids include sterols such as cholesterol and its derivatives, for example, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

[0325] In some embodiments, the non-cationic lipid present in the lipid particle (e.g., LNP) comprises or consists of cholesterol or its derivative, e.g., a phospholipid-free lipid particle formulation. In other embodiments, the non-cationic lipid present in the lipid particle (e.g., LNP) comprises or consists of one or more phospholipids, e.g., a cholesterol-free lipid particle formulation. In further embodiments, the non-cationic lipid present in the lipid particle (e.g., LNP) comprises or consists of a mixture of one or more phospholipids and cholesterol or its derivative.

[0326] Other examples of non-cationic lipids suitable for use in the present invention include, for example, phosphorus-free lipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymer, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethoxylated fatty acid amide, dioctadecyldimethylammonium bromide, ceramide, sphingomyelin, and the like.

[0327] In some embodiments, the non-cationic lipid can 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 particle.

[0328] In certain embodiments, the cholesterol present in the phospholipid-free lipid 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. By way of non-limiting example, the phospholipid-free lipid particles can contain about 37 mol% cholesterol of the total lipids present in the particles.

[0329] In certain other embodiments, the cholesterol present in the lipid particles containing a mixture of phospholipid 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. By way of non-limiting example, the lipid particles containing a mixture of phospholipid and cholesterol can contain about 34 mol% cholesterol of the total lipids present in the particles.

[0330] In a further embodiment, the cholesterol present in the lipid particles containing a mixture of phospholipid 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. By way of non-limiting example, the lipid particles containing a mixture of phospholipid and cholesterol can contain about 20 mol% cholesterol of the total lipids present in the particles.

[0331] In embodiments where the lipid particles contain a mixture of phospholipid and cholesterol or a cholesterol derivative, 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 phospholipid and cholesterol may contain a phospholipid such as DPPC or DSPC at about 7 mol% of the total lipids present in the particles (e.g., 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 phospholipid and cholesterol may contain a phospholipid such as DPPC or DSPC at about 20 mol% of the total lipids present in the particles (e.g., in a mixture with about 20 mol% cholesterol).

[0332] Lipid conjugate In addition to cationic and non-cationic lipids, the lipid particles (e.g., LNPs) of the present invention contain lipid conjugates. Conjugate lipids are useful in that they inhibit aggregation of the particles. Suitable conjugate 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 contain either a PEG-lipid conjugate or an ATTA-lipid conjugate together with CPL.

[0333] In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examples of PEG-lipids include, for example, PEG coupled to dialkyloxypropyl as described in PCT Publication No. 05 / 026372 (PEG-DAA), PEG coupled to diacylglycerol as described in, for example, U.S. Patent Application Publication Nos. 20030077829 and 2005008689 (PEG-DAG), PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramide as described in, for example, U.S. Patent No. 5,885,613, PEG conjugated to cholesterol or its derivatives, and mixtures thereof, but are not limited thereto. The disclosures of these patent documents are hereby incorporated 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.

[0334] PEG is a linear water-soluble polymer of ethylene glycol repeating units with two terminal hydroxyl groups. PEG is classified by its molecular weight. For example, PEG2000 has an average molecular weight of about 2,000 Daltons, and PEG5000 has an average molecular weight of about 5,000 Daltons. PEG is commercially available from Sigma Chemical Co. and other companies, and includes, for example, the following: methoxypolyethylene glycol (MePEG-OH), methoxypolyethylene glycol-succinate (MePEG-S), methoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), methoxypolyethylene glycol-amine (MePEG-NH2), methoxypolyethylene glycol-tresylate (MePEG-TRES), and methoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). Other PEGs (e.g., mPEG(20KDa) amine) such as those described in U.S. Patent Nos. 6,774,180 and 7,053,150 are also useful for the preparation of the PEG-lipid conjugates of the present invention. The disclosures of these patents are hereby incorporated by reference in their entirety for all purposes. Further, methoxypolyethylene glycol acetic acid (MePEG-CH2COOH) is particularly useful for the preparation of PEG-lipid conjugates, including, for example, PEG-DAA conjugates.

[0335] The PEG moiety of the PEG-lipid conjugates described herein can include an average molecular weight in the range of about 550 Daltons to about 10,000 Daltons. In certain cases, the PEG moiety 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 a preferred embodiment, the PEG moiety has an average molecular weight of about 2,000 Daltons or about 750 Daltons.

[0336] In certain cases, the PEG may be optionally substituted by an alkyl group, an alkoxy group, an acyl group, or an aryl group. The PEG may be directly conjugated to the lipid or linked to the lipid via a linker moiety. For example, any linker moiety suitable for coupling PEG to a lipid can be used, including linker moieties that contain an ester-free linker moiety and an ester-containing linker moiety. In a preferred embodiment, 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, amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, disulfide, and combinations thereof (e.g., a linker containing both a carbamate linker moiety and an amide linker moiety). In a preferred embodiment, the carbamate linker is used to couple PEG to the lipid.

[0337] In other embodiments, an ester-containing linker moiety is used to couple PEG to the lipid. Suitable ester-containing linker moieties include, for example, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), sulfonate ester, and combinations thereof.

[0338] Further PEG-lipid conjugates suitable for use in the present invention include, but are not limited to, the formula:

Chemical formula

[0339] Conjugate lipids can include, for example, PEG-lipids including 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, 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 substituted with one or more anion precursor groups, and 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 can be A-PEG-dilauryl oxypropyl (C12), A-PEG-dimyristyl oxypropyl (C14), A-PEG-dipalmityl oxypropyl (C16), or A-PEG-distearyl oxypropyl (C18), or mixtures thereof.

[0340] Phosphatidylethanolamines having various acyl chain groups of various chain lengths and degrees of saturation can be conjugated to PEG to form lipid conjugates. Such phosphatidylethanolamines can be commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. C 10 ~C 20 Phosphatidylethanolamines containing saturated or unsaturated fatty acids having a carbon chain length in the range of are preferred. Phosphatidylethanolamines having mono- or di-unsaturated 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), dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0341] The term "ATTR" or "polyamide" refers, without limitation, to the compounds described in U.S. Patent Nos. 6,320,017 and 6,586,559 (the disclosures of which are incorporated herein by reference in their entirety for all purposes). These compounds include the formula:

Chemical formula

[0342] The term "diacylglycerol" refers to a compound having two fatty acyl chains R 1 and R 2 which are both independently bonded to the 1- and 2-positions of glycerol by ester bonds and have from 2 to 30 carbons. The acyl groups may be saturated or may have various degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauryl (C 12 ), myristyl (C 14 ), palmityl (C 16 ), stearyl (C 18 ), and icosyl (C 20 ). In a preferred embodiment, R 1 and R 2 are the same, for example, R 1 and R 2 are both myristyl (e.g., dimyristyl), R 1 and R 2 are both stearyl (e.g., distearyl), etc. Diacylglycerol has the following general formula:

Chemical formula

[0343] The term "dialkyloxypropyl" refers to a compound having two alkyl chains R 1 and R 2 which are both independently having from 2 to 30 carbons. The alkyl groups may be saturated or may have various degrees of unsaturation. Dialkyloxypropyl has the following general formula:

Chemical formula

[0344] In a preferred embodiment, the PEG-lipid is of the following formula:

Chemical formula

[0345] In the above formula VII, PEG has an average molecular weight in the range of about 550 Daltons to about 10,000 Daltons. In certain 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 a preferred embodiment, 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. In certain embodiments, the terminal hydroxyl group is substituted with a methoxy group or a methyl group.

[0346] In a preferred embodiment, "L" is an ester-free linker moiety. Suitable ester-free linkers include, but are not limited to, an amide linker moiety, an amino linker moiety, a carbonyl linker moiety, a carbamate linker moiety, a urea linker moiety, an ether linker moiety, a disulfide linker moiety, a succinimidyl linker moiety, and combinations thereof. In a preferred embodiment, the ester-free linker moiety is a carbamate linker moiety (e.g., PEG-C-DAA conjugate). In another preferred embodiment, the ester-free linker moiety is an amide linker moiety (e.g., PEG-A-DAA conjugate). In yet another preferred embodiment, the ester-free linker moiety is a succinimidyl linker moiety (e.g., PEG-S-DAA conjugate).

[0347] In certain embodiments, the PEG-lipid conjugate is selected from:

Chemical formula

[0348] 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 linkages. Those skilled in the art will recognize that methods and reagents for forming these linkages are well-known and readily available. See, for example, March, ADVANCED ORGANIC CHEMISTRY (Wiley 1992), Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989), and Furniss, VOGEL’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 the PEG-DAA conjugate. Those skilled in the art will recognize that such techniques are well-known. See, for example, Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991).

[0349] Preferably, the PEG-DAA conjugate is a dilauroxypropyl (C 12 )-PEG conjugate, a dimyristyloxypropyl (C 14 )-PEG conjugate, a dipalmityloxypropyl (C 16 )-PEG conjugate, or a distearyloxypropyl (C 18 )-PEG conjugate. Those skilled in the art will readily understand that other dialkyloxypropyls can be used in the PEG-DAA conjugates of the present invention.

[0350] In addition to the foregoing, it will be readily apparent to those skilled in the art that other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0351] The charge of the polycationic moiety can be distributed over the entire particle moiety or, alternatively, can be a different concentration of charge density, e.g., a charge spike, in a particular region of the particle moiety. If the charge density is distributed over the particle, the charge density can be distributed uniformly or non-uniformly. All variations of the charge distribution of the polycationic moiety are encompassed by the present invention.

[0352] Lipid "A" and non-immunogenic polymer "W" can be linked by various methods, preferably by covalent bonds. Methods known to those skilled in the art can be used for the covalent bonding of "A" and "W". Suitable linkages include, but are not limited to, amide bonds, amine bonds, carboxyl bonds, carbonate bonds, carbamate bonds, ester bonds, and hydrazone bonds. It will be apparent to those skilled in the art that "A" and "W" must have complementary functional groups in order to effect the linkage. The reaction of these two groups, one on the lipid and the other on the polymer, results in the desired linkage. For example, when the lipid is diacylglycerol and the terminal hydroxyl is activated with, for example, NHS and DCC to form an active ester, and then this lipid reacts with a polymer containing an amino group such as a polyamide (see, e.g., U.S. Patent Nos. 6,320,017 and 6,586,559. These disclosures are hereby incorporated by reference in their entirety for all purposes), an amide bond is formed between the two groups.

[0353] In certain cases, ligands such as targeting ligands or chelating moieties for complexing calcium can be attached to the polycationic moiety. Preferably, after the ligand is attached, the cationic moiety maintains a positive charge. In certain cases, the attached ligand has a positive charge. Suitable ligands include, but are not limited to, compounds or devices having reactive functional groups, lipids, amphiphilic lipids, carrier compounds, biocompatible compounds, biomaterials, biopolymers, medical devices, analytically detectable compounds, therapeutically active compounds, enzymes, peptides, proteins, antibodies, immunostimulants, radiolabels, fluorescent emitters, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, other targeting moieties, or toxins.

[0354] The lipid conjugate (e.g., PEG-lipid) typically constitutes about 0.1 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 1 mol% to about 10 mol%, about 0.6 mol% to about 1.9 mol%, about 0.7 mol% to about 1.8 mol%, about 0.8 mol% to about 1.7 mol%, about 0.9 mol% to about 1.6 mol%, about 0.9 mol% to about 1.8 mol%, about 1 mol% to about 1.8 mol%, about 1 mol% to about 1.7 mol%, about 1.2 mol% to about 1.8 mol%, about 1.2 mol% to about 1.7 mol%, about 1.3 mol% to about 1.6 mol%, or about 1.4 mol% to about 1.5 mol% of the total lipids present in the particles.

[0355] One of ordinary skill 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 fusogenic.

[0356] By controlling the composition and concentration of the lipid conjugate, the rate at which the lipid conjugate exchanges with the outside of the nucleic acid-lipid particle and, in turn, the rate at which the nucleic acid-lipid particle becomes membrane fusogenic can be controlled. For example, when a PEG-phosphatidylethanolamine conjugate or a PEG-ceramide conjugate is used as the lipid conjugate, the rate at which the nucleic acid-lipid particle becomes membrane fusogenic can be varied, for example, by changing the concentration of the lipid conjugate, by changing the molecular weight of the PEG, or by changing the chain length and degree of saturation of the acyl chain groups on phosphatidylethanolamine or ceramide. Further, other variables, including, for example, pH, temperature, ionic strength, etc., can be used to vary and / or control the rate at which the nucleic acid-lipid particle becomes membrane fusogenic. Other methods that can be used to control the rate at which the nucleic acid-lipid particle becomes membrane fusogenic will be apparent to those skilled in the art upon reading the present disclosure.

[0357] Preparation of Lipid Particles The lipid particles of the present invention, such as LNPs, in which an active agent 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.

[0358] In preferred embodiments, the cationic lipid is a lipid of formula I, II, and III, or a combination thereof. In other preferred embodiments, the non-cationic lipid is egg sphingomyelin (ESM), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dipalmitoyl-phosphatidylcholine (DPPC), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, 14:0 PE (1,2-dimyristoyl-phosphatidylethanolamine (DMPE)), 16:0 PE (1,2-dipalmitoyl-phosphatidylethanolamine (DPPE)), 18:0 PE (1,2-distearoyl-phosphatidylethanolamine (DSPE)), 18:1 PE (1,2-dioleoyl-phosphatidylethanolamine (DOPE)), 18:1 trans PE (1,2-delaidoyl-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-based polymers (e.g., PEG2000, PEG5000, PEG-modified diacylglycerol, or PEG-modified dialkyloxypropyl), cholesterol, or a combination thereof.

[0359] In certain embodiments, the present invention provides an LNP produced by a process that includes a continuous mixing method, e.g., 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 encapsulating the nucleic acid (e.g., interfering RNA or mRNA). This process and the apparatus for carrying out this process are described in detail in U.S. Patent Application Publication No. 20040142025, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0360] By the operation of continuously introducing a lipid and a buffer solution into a mixing environment such as a mixing chamber, continuous dilution of the lipid solution by the buffer solution occurs, whereby liposomes are produced substantially instantaneously upon mixing. As used herein, the phrase (and variations) "continuously diluting a lipid solution with a buffer solution" generally means that the lipid solution is diluted sufficiently rapidly with sufficient force to result in the formation of vesicles during the hydration process. By mixing an aqueous solution containing nucleic acid with an organic lipid solution, the organic lipid solution undergoes continuous stepwise dilution in the presence of a buffer solution (i.e., an aqueous solution) to produce nucleic acid-lipid particles.

[0361] LNPs formed using the continuous mixing method typically have a size 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. The particles thus formed do not aggregate and are optionally sized to obtain a uniform particle diameter.

[0362] In another embodiment, the present invention provides LNPs produced by a direct dilution process that includes forming a liposome solution and immediately directly introducing the liposome solution into a collection container containing a controlled amount of dilution buffer. In a preferred embodiment, the collection container includes one or more components configured to stir 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 the liposome solution introduced therein. As a non-limiting example, a liposome solution in 45% ethanol, when introduced into a collection container containing an equal volume of dilution buffer, will advantageously result in smaller particles.

[0363] In yet another embodiment, the present invention provides LNPs produced by a direct dilution process that fluidly couples a third reservoir containing a dilution buffer to a second mixing region. In this embodiment, the liposome solution formed in the first mixing region is immediately directly mixed with the dilution buffer in the second mixing region. In a preferred embodiment, the second mixing region includes a T-connector arranged such that the flows of the liposome solution and the dilution buffer converge as 180° opposing flows, although connectors that provide a shallower angle, such as from 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 thereto from the first mixing region. This embodiment advantageously allows for further control of the flow of the dilution buffer mixing with the liposome solution in the second mixing region, and thus also of the concentration of the liposome solution in the buffer throughout the second mixing process. Such control of the flow rate of the dilution buffer advantageously enables the formation of small particle sizes at reduced concentrations.

[0364] These processes and the apparatus for carrying out these direct dilution processes are described in detail in U.S. Patent Application Publication No. 20070042031, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0365] LNPs formed using the direct dilution process typically have a size of from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, or from about 70 nm to about 90 nm. Particles formed in this manner do not aggregate and are optionally sized to obtain a uniform particle size.

[0366] If desired, the lipid particles (e.g., LNPs) of the present invention can be sized by any method available for sizing liposomes. Sizing can be carried out to obtain a desired size range and a relatively narrow particle size distribution.

[0367] Several techniques are available for sizing particles to a desired size. One sizing method used for liposomes and equally applicable to the particles of the present invention is described in U.S. Patent No. 4,737,323, the disclosure of which is incorporated herein by reference in its entirety for all purposes. By subjecting the particle suspension to sonication, either bath sonication or probe sonication, the size is gradually reduced to particles of a size less 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 from about 60 to about 80 nm) is observed. In both methods, the particle size distribution can be monitored by conventional laser light particle sizing or QELS.

[0368] Extrusion of particles through a porous polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing the particle size to a relatively well-defined size distribution. Typically, the suspension is circulated through the membrane one or more times until the desired particle size distribution is obtained. The particles can be sequentially passed through membranes with smaller pores for extrusion to gradually reduce the size.

[0369] In some embodiments, the nucleic acid in the LNP is pre-concentrated as described, for example, in U.S. Patent Application No. 09 / 744,103, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0370] In other embodiments, the method further comprises adding a non-lipid polycation useful for performing lipofection of cells using the compositions of the present invention. Examples of suitable non-lipid polycations include hexadimethrine bromide (sold under the trade name POLYBRENE® by Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts of hexadimethrine. 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 are formed.

[0371] In some embodiments, the nucleic acid to lipid ratio (mass / mass ratio) in the formed LNP ranges from about 0.01 to about 0.2, from about 0.02 to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about 0.08. The ratio of 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 corresponding to about 1.25 mg of total lipid per 50 μg of nucleic acid, more preferably in the range of about 0.01 to about 0.08. In other preferred embodiments, the particles have a nucleic acid:lipid mass ratio of about 0.08.

[0372] In other embodiments, the lipid-to-nucleic acid ratio (mass / mass ratio) in the formed LNPs ranges from about 1 (1:1) to about 100 (100:1), from about 5 (5:1) to about 100 (100:1), from about 1 (1:1) to about 50 (50:1), from about 2 (2:1) to about 50 (50:1), from about 3 (3:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 5 (5:1) to about 50 (50:1), from about 1 (1:1) to about 25 (25:1), from about 2 (2:1) to about 25 (25:1), from about 3 (3:1) to about 25 (25:1), from about 4 (4:1) to about 25 (25:1), from about 5 (5:1) to about 25 (25:1), from about 5 (5:1) to about 20 (20:1), from about 5 (5:1) to about 15 (15:1), from about 5 (5:1) to about 10 (10:1), about 5 (5:1), 6 (6:1), 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), 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 ratio of starting materials also typically falls within this range.

[0373] As described above, the conjugate lipid may further contain CPL. Various general methods for making LNP-CPL (CPL-containing LNP) are discussed herein. Two general techniques include the "post-insertion" technique, i.e., insertion of CPL into, for example, pre-formed LNP, and the "standard" technique of including CPL in the lipid mixture, for example, during the LNP formation process. The post-insertion technique mainly results in LNP having CPL on the outer surface of the LNP bilayer membrane, while the standard technique yields LNP having CPL on both the inner and outer surfaces. This method is particularly useful for vesicles made from phospholipids (which may contain cholesterol) and is also useful for vesicles containing PEG-lipids (e.g., PEG-DAA and PEG-DAG). Methods for making LNP-CPL are taught, for example, in U.S. Pat. Nos. 5,705,385, 6,586,410, 5,981,501, 6,534,484, and 6,852,334, U.S. Patent Application Publication No. 20020072121, and PCT Publication No. 00 / 62813, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

[0374] Other methods for making LNP can be found, for example, in U.S. Pat. No. 9,005,654 and PCT Publication No. 2007 / 012191, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

[0375] Kit The invention also provides lipid particles (e.g., LNP) in kit form. The kit can include a container compartmentalized to hold the various components of the lipid particles (e.g., an active or therapeutic agent such as a nucleic acid and the individual lipid components of the particle). In some embodiments, the kit can further include an endosome membrane destabilizing agent (e.g., calcium ions). The kit typically includes the lipid particle composition of the invention, preferably in dehydrated form, together with instructions for its rehydration and administration.

[0376] As described herein, the lipid particles of the present invention (e.g., LNP) can be adapted to preferentially target a specific tissue, organ, or tumor of interest. In certain cases, preferential targeting of lipid particles such as LNP 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 on the other hand, it has been found that a 1:57 PEG-cDMA LNP formulation can be used to preferentially target the liver (including liver tumors).

[0377] In certain other cases, it may be desirable to have a targeting moiety attached to the surface of the lipid particle in order to further improve the targeting of the particle. Methods of attaching a targeting moiety (e.g., an antibody, protein, etc.) to a lipid (such as those used in the particles of the present invention) are known to those skilled in the art.

[0378] Administration of Lipid Particles The lipid particles of the present invention (e.g., LNP) are useful for introducing an active or therapeutic agent (e.g., a nucleic acid such as an interfering RNA or mRNA) into cells after they are formed. Accordingly, the present invention also provides a method for introducing an active or therapeutic agent such as a nucleic acid (e.g., an interfering RNA or mRNA) into cells. This method is carried out in vitro or in vivo by first forming the particles as described above and then contacting the particles with the cells for a period of time sufficient for delivery of the active or therapeutic agent to the cells.

[0379] The lipid particles of the present invention (e.g., LNP) can be adsorbed to almost all cell types with which they are mixed or contacted. Once adsorbed, the particles can either be endocytosed by a portion of the cell, exchange lipids with the cell membrane, or fuse with the cell. Introduction or incorporation of the active or therapeutic agent (e.g., nucleic acid) portion of the particle can occur via any one of these pathways. In particular, when fusion occurs, the particle membrane is incorporated into the cell membrane and the contents of the particle mix with the intracellular fluid.

[0380] The lipid particles of the present invention (e.g., LNP) can be administered either alone or as a mixture with a pharmaceutically acceptable carrier (e.g., physiological saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practice. Generally, buffered physiological saline (e.g., 135 - 150 mM NaCl) will be used as the pharmaceutically acceptable carrier. The lipids can also be frozen for stabilization. For example, the lipids can be stored at -20°C under high salt concentration (e.g., 500 mM NaCl) in Tris buffer at pH 8. As a further example, the lipids may be stored at -80°C in Tris buffer at pH 8 containing a mixture of sucrose and maltose. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, etc., including glycoproteins for enhancing stability such as albumin, lipoproteins, globulins. Further suitable carriers are described, for example, in REMINGTON’S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coating agents, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans.

[0381] The pharmaceutically acceptable carrier is generally added after the formation of the lipid particles. Thus, after particle formation, the particles can be diluted in a pharmaceutically acceptable carrier such as physiological buffered saline.

[0382] The concentration of particles in a pharmaceutical preparation can vary widely, for example, from less than about 0.05% by weight, usually about 2 - 5% by weight or at least about 2 - 5% by weight, to as high as about 10 - 90% by weight, and will be selected primarily according to the particular mode of administration, mainly by the 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 or severe hypertension associated with atherosclerosis. Alternatively, particles composed of irritating lipids may be diluted to a low concentration to reduce inflammation at the administration site.

[0383] 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 and lyophilized under aseptic conditions, and the lyophilized preparation may be mixed with a sterile aqueous solution before administration. The composition may contain pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, osmotic pressure adjusters, etc. required to approximate physiological conditions, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride. Further, the particle suspension may contain a lipid protecting agent that protects the lipid from damage due to free radicals and lipid peroxidation during storage. Lipophilic free radical quenchers such as alpha tocopherol and water-soluble iron-specific chelating agents such as deferoxamine are suitable.

[0384] in vivo administration Systemic delivery for in vivo therapy, for example, delivery of therapeutic nucleic acids to distal target cells via body systems such as circulation, is achieved using nucleic acid-lipid particles such as those described in PCT Publications WO05 / 007196, WO05 / 121348, WO05 / 120152, and WO04 / 002453 (the disclosures of which are hereby incorporated 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, are small in size, and are suitable for repeated dosing.

[0385] In the case of in vivo administration, the administration can be by any method known in the art, such as injection, oral administration, inhalation (e.g., intranasal or intratracheal), transdermal application, or rectal administration. The administration can be carried out as a single dose or in divided doses. The pharmaceutical composition can be administered parenterally, e.g., into a joint, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the pharmaceutical composition is administered intravenously or intraperitoneally by bolus injection (see, e.g., 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). Still other methods of administering lipid-based therapeutic agents are described, e.g., in U.S. Patent Nos. 3,993,754, 4,145,410, 4,235,871, 4,224,179, 4,522,803, and 4,588,578. The lipid particles can be administered by direct injection at the site of the disease or by injection at a site distal to the site of the disease (see, e.g., Culver, HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp. 70-71 (1994)). The disclosures of the foregoing references are incorporated herein by reference in their entirety for all purposes.

[0386] The compositions of the invention can be formulated into aerosol preparations, either alone or in combination with other suitable ingredients, for administration via inhalation (e.g., intranasal or intratracheal) (e.g., they can be "sprayed") (see Brigham et al., Am. J. Sci., 298:278 (1989)). The aerosol preparations can be placed in a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen, etc.

[0387] In certain embodiments, the pharmaceutical composition can be delivered by intranasal spray, inhalation, and / or other aerosol delivery vehicles. Methods for delivering nucleic acid compositions directly to the lungs by nasal aerosol spray are described, for example, in U.S. Pat. Nos. 5,756,353 and 5,804,212. Similarly, drug delivery using intranasal microparticle resins and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) is also well known in the pharmaceutical art. Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is described in U.S. Pat. No. 5,780,045. The disclosures of the foregoing patents are hereby incorporated by reference in their entirety for all purposes.

[0388] For example, formulations suitable for parenteral administration by, for example, intra-articular (into the joint), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, aqueous and non-aqueous isotonic sterile injection solutions, and aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. In the practice of the present invention, the compositions are preferably administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally.

[0389] Generally, for intravenous administration, the lipid particle formulation is 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, such as water, buffered water, 0.4% saline, 0.3% glycine, etc. can be used, and these aqueous carriers can contain glycoproteins for enhancing stability, such as albumin, lipoproteins, globulins, etc. Generally, buffered physiological saline (135 - 150 mM NaCl) is used as the pharmaceutically acceptable carrier, but other suitable carriers will also suffice. These compositions can be sterilized by conventional liposome sterilization techniques such as filtration. The compositions may contain pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, osmotic pressure adjusters, wetting agents, etc. required to approximate physiological conditions, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. These compositions may be sterilized using the aforementioned techniques, or alternatively, produced under sterile conditions. The resulting aqueous solution may be packaged for use, or filtered under sterile conditions and lyophilized, and the lyophilized preparation may be mixed with a sterile aqueous solution prior to administration.

[0390] In 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, troches, capsules, pills, lozenges, elixirs, mouthwashes, suspensions, oral sprays, syrups, oblatum, etc. (see, for example, U.S. Patent Nos. 5,641,515, 5,580,579, and 5,792,451. These disclosures are hereby incorporated by reference in their entirety for all purposes). These oral dosage forms may also contain, hereinafter, i.e., binders, gelatin; excipients, lubricants, and / or flavoring agents. When 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 modify the physical form of the dosage unit. Of course, any substance used in the preparation of any unit dosage form should be pharmaceutically pure and substantially non-toxic in the amounts used.

[0391] Typically, these oral formulations can contain at least about 0.1% or more lipid particles, although the percentage of particles can of course vary and, conveniently, can 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 manner that a suitable dosage is obtained for 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 contemplated by those skilled in the art in preparing such pharmaceutical formulations, and thus, various dosages and treatment regimens may be desirable.

[0392] Formulations suitable for oral administration include, for example: (a) liquid solutions, such as a nucleic acid (e.g., interfering RNA or mRNA) and other therapeutically effective amounts of encapsulated therapeutics suspended in a diluent such as water, saline, or PEG400; (b) capsules, sachets, or tablets each containing a therapeutically effective amount of a nucleic acid (e.g., interfering RNA or mRNA) and other therapeutics in the form of a liquid, solid, granule, or gelatin, respectively; (c) suspensions in suitable liquids; and (d) suitable emulsions. Tablet forms may include lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and one or more of other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and pharmaceutically acceptable carriers. Lozenge forms may include gelatin and glycerin containing a flavoring agent, such as a nucleic acid (e.g., interfering RNA or mRNA) and other therapeutics in sucrose, and carriers known in the art in addition to the therapeutics, or pastilles containing the therapeutics in an inert base such as sucrose and gum arabic emulsion, gel, and the like.

[0393] In another example of their use, lipid particles can be incorporated into a wide range of topical dosage forms. For example, suspensions containing nucleic acid-lipid particles such as LNP can be formulated and administered as gels, oils, emulsions, topical creams, pastes, ointments, lotions, foams, mousses, and the like.

[0394] When preparing a pharmaceutical preparation of the lipid particles of the present invention, it is preferred to use a large amount of particles that have been purified to reduce or remove empty particles or particles with therapeutics such as nucleic acids associated with the outer surface.

[0395] The methods of the present invention can be practiced in a variety of hosts. Preferred hosts include mammalian species such as primates (e.g., humans, chimpanzees, and other non-human primates), dogs, cats, horses, cows, sheep, goats, rodents (e.g., rats and mice), rabbits, and pigs.

[0396] 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 age, weight, and condition of the patient, as well as the judgment of the clinician, but generally ranges from about 0.01 to about 50 mg / kg body weight, preferably from about 0.1 to about 5 mg / kg body weight, or about 10 8 ~10 10 particles per administration (e.g., injection).

[0397] in vitro administration For in vitro use, delivery of a therapeutic agent such as a nucleic acid (e.g., interfering RNA or mRNA) can be performed on any cell grown in culture, regardless of whether it is of plant origin or animal origin, vertebrate or invertebrate, and of any tissue or type. In a preferred embodiment, the cell is an animal cell, more preferably a mammalian cell, and most preferably a human cell.

[0398] When performed in vitro, the contact between the cell and the lipid particles is carried out in a biologically compatible medium. The concentration of the particles varies widely depending on the particular application, but generally ranges from about 1 μmol to about 10 mmol. Treatment of cells with lipid particles is generally carried out at physiological temperature (about 37 °C) for a period of about 1 to 48 hours, preferably about 2 to 4 hours.

[0399] In one group of preferred embodiments, the lipid particle suspension is added to cells plated at 60 - 80% confluence with a cell density of about 10 3 ~ about 10 5 cells / ml, more preferably about 2 × 10 4 cells / ml. The concentration of the suspension added to the cells is preferably about 0.01 to 0.2 μg / ml, more preferably about 0.1 μg / ml.

[0400] The delivery efficiency of the LNPs or other lipid particles of the present invention can be optimized using the Endosomal Release Parameter (ERP) assay. The ERP assay is described in detail in U.S. Patent Application Publication No. 20030077829, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. More specifically, the purpose of the ERP assay is to discriminate the effects of the various cationic lipid and helper lipid components of the LNP based on their relative effects on endosomal membrane binding / uptake or fusion / destabilization with the endosomal membrane. This assay enables the quantitative determination of how each component of the LNP or other lipid particle affects the delivery efficiency, thereby enabling the optimization of the LNP or other lipid particle. 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, the ERP assay 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, the ERP assay can be adapted to measure the expression of a target protein in the presence or absence of mRNA. By comparing the ERPs for each of the various LNPs or other lipid particles, an optimized system, e.g., the LNP or other lipid particle that is most highly taken up intracellularly, can be readily determined.

[0401] Cells for Delivery of Lipid Particles The compositions and methods of the present invention are used for treating 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 and smooth muscle cells, osteoblasts, neurons, resting lymphocytes, terminally differentiated cells, noncycling primary cells, parenchymal cells, lymphoid cells, epithelial cells, bone cells, and the like. In a preferred embodiment, an active 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, cholangiocarcinoma 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, renal 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 blood cancer cells.

[0402] 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. The methods and compositions can be used in cells of a wide variety of vertebrates, including mammals such as dogs, cats, horses, cows, sheep, goats, rodents (e.g., mice, rats, and guinea pigs), rabbits, pigs, and primates (e.g., monkeys, chimpanzees, and humans).

[0403] Within the range that may be required, tissue culture of cells 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 lines are often in the form of monolayer cells, but cell suspensions are also used.

[0404] Detection of lipid particles In some embodiments, the lipid particles of the present invention (e.g., LNP) are detectable in a subject at about 1, 2, 3, 4, 5, 6, 7, 8 hours or more time points. In other embodiments, the lipid particles of the present invention (e.g., LNP) are detectable in a subject about 8, 12, 24, 48, 60, 72, or 96 hours after administration of the particles, or about 6, 8, 10, 12, 14, 16, 18, 19, 22, 24, 25, or 28 days later. 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 a therapeutic nucleic acid such as interfering RNA (e.g., siRNA) or mRNA sequence, by detection of the target sequence of interest (i.e., by detecting the expression or reduction of expression of the target sequence), or by a combination thereof.

[0405] Detection of particles The lipid particles of the present invention, such as LNPs, can be detected using any method known in the art. For example, using methods well-known in the art, labels can be directly or indirectly coupled to the components of the lipid particles. Depending on the required sensitivity, ease of conjugation with lipid particle components, stability requirements, and available measurement means and disposal provisions, labels can be selected and a wide variety of labels can be used. 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 radioactive labels such as 33 P; enzymes such as horseradish peroxidase, alkaline phosphatase; and spectrocolorimetric labels such as colloidal gold or plastic beads such as colored glass or polystyrene, polypropylene, latex, etc., but are not limited thereto. Labels can be detected using any means known in the art.

[0406] Detection of Nucleic Acids Nucleic acids (e.g., interfering RNA or mRNA) are detected and quantified by any of several means well-known to those skilled in the art herein. Detection of nucleic acids can be performed by well-known methods such as Southern analysis, Northern analysis, gel electrophoresis, PCR, radioactive labeling, scintillation counting, and affinity chromatography. Additional analytical biochemical methods such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography can also be used.

[0407] The choice of nucleic acid hybridization format is not critical. A variety of nucleic acid hybridization formats are known to those of skill in the art. For example, common formats include sandwich assays and competitive or displacement assays. Hybridization techniques are generally described, for example, in “Nucleic Acid Hybridization, A Practical Approach,” Eds. Hames and Higgins, IRL Press (1985).

[0408] The sensitivity of a hybridization assay can be improved through the use of a nucleic acid amplification system that increases the target nucleic acid to be detected. In vitro amplification techniques suitable for the amplification of sequences for use as molecular probes or for the generation of nucleic acid fragments for subsequent subcloning are known.Examples of techniques sufficient to guide a practitioner 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 (trademark)), are found 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., 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., Gene, 89:117 (1990), and Sooknanan and Malek, Biotechnology, 13:563 (1995). Improved methods for cloning in vitro amplified nucleic acids are described in U.S. Patent No. 5,426,039.Other methods described in the art are nucleic acid sequence-based amplification (NASBA™, Cangene, Mississauga, Ontario) and the Qβ-replicase system. These systems can be used to directly identify mutants when designed such that PCR or LCR primers will only extend or ligate if the selected sequence is present. Alternatively, the selected sequence can generally be amplified, for example using non-specific PCR primers, and then the amplified target region can be searched for specific sequences that indicate a mutation. The disclosures of the foregoing references are hereby incorporated by reference in their entirety for all purposes.

[0409] For example, nucleic acids for use as a probe in an in vitro amplification method, for use as a gene probe, or as an inhibitor component are usually chemically synthesized according to the solid phase phosphoramidite triester method described by Beaucage et al., Tetrahedron Letts., 22:1859-1862 (1981), using, for example, an automated synthesizer as described by Needham VanDevanter et al., Nucleic Acids Res., 12:6159 (1984). If necessary, purification of the polynucleotide is usually carried out either by denaturing acrylamide gel electrophoresis or by anion exchange HPLC as described by Pearson et al., J. Chrom., 255:137-149 (1983). The sequence of the synthetic polynucleotide can be verified using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology, 65:499.

[0410] An alternative means for measuring the transcription level is in situ hybridization. In situ hybridization assays are well known and are generally described in Angerer et al., Methods Enzymol., 152:649 (1987). In an in situ hybridization assay, cells are fixed to a solid support, usually a slide glass. When searching for DNA, the cells are denatured with heat or alkali. Next, the cells are contacted with a hybridization solution at a moderate temperature to anneal a labeled specific probe. The probe is preferably labeled with a radioisotope or a fluorescent reporter.

Example

[0411] Example The present invention will be described in more detail by specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present invention in any way. Those skilled in the art will readily recognize various non-essential parameters that can be changed or modified to yield essentially the same results.

[0412] Example 1

Chemical formula

[0413] Synthesis of (Z)-deca-4-en-1-yl methanesulfonate (1)

Chemical formula

[0414] Synthesis of (Z)-undec-5-enenitrile (2)

Chemical formula

[0415] Synthesis of ethyl (Z)-undec-5-enoate (3)

Chemical formula

[0416] Synthesis of (6Z,16Z)-12-hydroxydocosa-6,16-dien-11-one (4)

Chemical formula

[0417] Synthesis of (Z)-undeca-5-enoic acid (5)

Chemical formula

[0418] Synthesis of (6Z,16Z)-12-oxodocosa-6,16-dien-11-yl (Z)-undec-5-enoate (6)

Chemical formula

[0419] Synthesis of (6Z,16Z)-12-(11-oxidanelyl)docosa-6,16-dien-11-yl (Z)-undec-5-enoate (7) [Chemical formula] (5Z,15Z)-10-oxoheneicosa-5,15-dien-11-yl (5Z)-undeca-5-enoate 6 (3.5 g, 7.16 mmol) was added slowly in portions over 30 minutes to a cooled solution (0 °C) containing anhydrous methanol (20 mL, 0.36 M) and THF (10 mL, 0.72 M). After the solution was stirred at room temperature for 4 hours, the reaction was quenched slowly with water (5 mL) and the solution was concentrated. The remaining aqueous solution was diluted with water (50 mL) and then extracted with EtOAc (2×75 mL), dried over magnesium sulfate, filtered and concentrated to dryness. Purification by column chromatography (ethyl acetate in hexane concentration 0→50%) gave (5Z,15Z)-10-hydroxyheneicosa-5,15-dien-11-yl (5Z)-undeca-5-enoate 7 (2.1 g, 59.8%) as a colorless oil.

[0420] Synthesis of (6Z,16Z)-12-((6-bromohexanoyl)oxy)docosa-6,16-dien-11-yl (Z)-undeca-5-enoate (8)

Chemical formula

[0421] Synthesis of (6Z,16Z)-12-((6-(dimethylamino)hexanoyl)oxy)docosa-6,16-dien-11-yl (Z)-undec-5-enoate (9) [Chemical formula] A solution containing (6Z,16Z)-12-[(6-bromohexanoyl)oxy]docosa-6,16-dien-11-yl (5Z)-undec-5-enoate 8 (900 mg, 1.32 mmol) in ethanol (6.6 mL, 2 M, 13.2 mmol) containing dimethylamine was heated overnight at 90 °C in a sealed reaction vessel. At the completion point, the solution was poured into saturated sodium bicarbonate and stirred for 30 minutes. The solution was extracted with ethyl acetate (3 times with 75 mL), the extracts were combined, dried over magnesium sulfate, filtered and then concentrated to dryness. The residue was purified by column chromatography (MeOH concentration in CH2Cl2 0→10%) to give (6Z,16Z)-12-{[6-(dimethylamino)hexanoyl]oxy}docosa-6,16-dien-11-yl (5Z)-undec-5-enoate 9 (440 mg, 51.6%) as a pale yellow oil. 1 H NMR (400 MHz, chloroform-d) δ 5.45 - 5.23 (m, 6H), 5.04 - 4.93 (m, 2H), 2.47 - 2.21 (m, 12H), 2.46 - 2.26 (m, 12H), 1.73 - 1.46 (m, 10H), 1.43 - 1.18 (m, 25H), 0.88 (t, J = 6.7 Hz, 9H).

[0422]

Chemical Structure

[0423] Synthesis of Methyl (Z)-dodec-5-enoate (13)

Chemical Structure

[0424] Synthesis of (7Z,17Z)-13-((6-(dimethylamino)hexanoyl)oxy)tetracosa-7,17-dien-12-yl (Z)-dodec-5-enoate (14)

Chemical Structure

[0425] Synthesis of 12-((6-(dimethylamino)hexanoyl)oxy)docosan-11-yl undecanoate (15)

Chemical Structure

[0426]

Chem.

Chem.

[0427] Synthesis of (6Z,16Z)-12-oxodocosa-6,16-dien-11-yl 6-(dimethylamino)hexanoate (17)

Chemical formula

[0428] Synthesis of (6Z,16Z)-12-hydroxydocosa-6,16-dien-11-yl 6-(dimethylamino)hexanoate (18)

Chemical formula

[0429] Synthesis of (6Z,16Z)-12-((6-(dimethylamino)hexanoyl)oxy)docosa-6,16-dien-11-yl (9Z,12Z)-octadeca-9,12-dienoate 19

Chem.

[0430]

Chem.

[0431] Accordingly, certain embodiments of the present invention are directed to any one of the compounds shown herein (e.g., those shown in Example 1 and / or the following table) or a salt thereof. TIFF0007709981000032.tif200165TIFF0007709981000033.tif189165

[0432] Example 2 Experiment General formulation procedure: Four components were included in the lipid solution: PEG-conjugated lipid, ionizable lipid, cholesterol, and phospholipid (e.g., DSPC). Lipid stocks were prepared using the described lipid identities and molar ratios. siRNA was dissolved in 100 mM acetate buffer (pH 4) such that the weight ratio of total lipid to siRNA was about 10:1 to 20:1 to prepare lipid nanoparticles (LNP). Equal volumes of the lipid solution and nucleic acid solution were passed through a T-connector at a flow rate of 400 mL / min and mixed, and then diluted in PBS (pH 7.4). Next, ethanol was removed and the external 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. The determination of nucleic acid concentration was performed by RiboGreen assay. The determination of particle size and polydispersity was performed using a Malvern Nano Series Zetasizer.

[0433] Activity evaluation: Generally, the activity of HSC-LNP was measured by intravenously injecting the LNP formulation into female Balb / C mice (5 - 8 weeks old) at 0.025 mg / kg. The siRNA LNP stock was filtered and diluted to the required dosing concentration immediately before injection. Forty-eight hours after administration (end point), the animals were euthanized using a lethal dose of ketamine / xylazine. The left half of the liver lobe was collected and placed in 1.5 mL of RNALater and stored overnight at 2 - 8 °C. The next day, approximately 20 - 25 mg of liver pieces were homogenized and the liver solubilate was used in the QuantiGene assay to test the relative expression levels of mouse target mRNA and GAPDH. The data obtained from each animal were normalized by the liver weight used in the assay and then relativized by the GAPDH signal. The data are reported as the average knockdown % for each group (the gene expression rate and knockdown rate of the PBS control group are set as 100% and 0%, respectively).

[0434] Tolerance evaluation: Generally, LNP formulations were intravenously injected into female Balb / C mice (5 - 8 weeks old) at about 0.03 - 3 mg / kg to evaluate the tolerance of HSC-LNP. Immediately before injection, the siRNA LNP stock was filtered and diluted to the required dosing concentration. Two hours after treatment, a tail nick was made to collect blood, which was processed to obtain plasma (for cytokine analysis by ELISA). At the end 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 and clinical chemistry analysis).

[0435] Briefly, blood (about 300 μL) was collected into an EDTA microtainer tube, inverted 10 times to mix the K2EDTA whole blood sample, and stored rapidly at 4°C until same-day shipment for hematological analysis. The remaining blood volume (about 500 μL) was collected and placed into a serum separator tube, inverted 5 times to mix the SST sample, and then allowed to clot at room temperature for 1 - 1.5 hours. The blood sample was centrifuged to collect serum for clinical chemistry analysis.

[0436] In the experiment, the following compounds were used.

Chem.

Chem.

[0437] In the experiments and figures described herein, the following compounds were used as representative compounds of the present invention.

Chem.

[0438] The following siRNA sequences (5’→3’) were used: RELN: S: GGucucAAGccAcucGuuudTsdT AS: AAACGAGUGGCUUGAGACCdTsdT Legend: Uppercase: Unmodified nucleotide Lowercase: 2’-OMe modification s: PS (phosphorothioate linkage) TTR: S: uGCUCUAUAAACCGUguUAGC AS: UAACACgGUUUAuAGAgCAAG Legend: Uppercase: Modified ribonucleotide Lowercase: 2’-OMe modification

[0439] In summary, as demonstrated by the results shown in this specification, the lipids described in this specification exhibit several important qualities for the delivery of therapeutic agents to hepatic stellate cells, including the ability to deliver therapeutic agents to HSCs and good tolerance, for example, those that can be used for the treatment of liver fibrosis. Finally, the preferred embodiments of the present invention will be described item by item. [Embodiment 1] Compound of formula (I):

Chemical formula

Chem.

Claims

1. A compound of formula (I) or a salt thereof: 【Chemical 1】 [wherein, R １ is (C ５ -C ２５ ) alkyl, (C ５ -C ２５ ) alkenyl, or (C ５ -C ２５ ) alkynyl, R ２ is (C ５ -C ２５ ) alkyl, (C ５ ) alkenyl, or (C ２５ ) alkynyl, and ５ -C ２５ ). R ３ is (C ５ -C ２５ ) alkyl, (C ５ -C ２５ ) alkenyl, or (C ５ -C ２５ ) alkynyl, R ４ is (C ３ -C １５ )alkyl, (C ３ -C １５ )alkenyl, or (C ３ -C １５ )alkynyl, and the (C ３ -C １５ )alkyl, (C ３ -C １５ )alkenyl, or C ３ -C １５ )alkynyl is substituted by one or more groups independently selected from chloro, bromo, iodo, and -NR ａ R ｂ . R ａ and R ｂ are each independently optionally substituted by one or more groups selected from halo and hydroxy and may be selected from the group consisting of (C １ -C ６ )alkyl and H, or R ａ and R ｂ together with the nitrogen to which they are attached form a ring selected from the group consisting of aziridine, azetidine, pyrrolidine, piperidine, piperazine, morpholino, and thiomorpholino, and said ring is optionally substituted by one or more groups independently selected from (C １ -C ６ )alkyl].

2. R １ 、 R ２ and R ３ are each independently (C ５ -C ２０ )alkyl, (C ５ -C ２０ )alkenyl, or (C ５ -C ２０ )alkynyl, the compound or a salt thereof according to claim 1.

3. R １ , R ２ and R ３ are each independently (C １０ -C ２０ )alkyl, (C １０ -C ２０ )alkenyl, or (C １０ -C ２０ )alkynyl, the compound or a salt thereof according to claim 1.

4. R １ is 4 - decen - 1 - yl or 8,10 - heptadecadien - 1 - yl; and / or R ２ is 4-decen-1-yl; and / or R ３ is 4-decen-1-yl The compound or a salt thereof according to claim 1.

5. R ４ is substituted by one or more groups independently selected from chloro, bromo, iodo, and -NR ａ R ｂ and is (C ３ -C １５ )alkyl, a compound according to any one of claims 1 to 4 or a salt thereof.

6. R ４ is substituted by one or more groups independently selected from chloro, bromo, iodo, and -NR ａ R ｂ and is (C ３ -C １０ )alkyl, the compound or a salt thereof according to any one of claims 1 to 4.

7. R ａ and R ｂ are each independently selected from the group consisting of (C １ -C ６ ) alkyl, the compound according to any one of claims 1 to 6, or a salt thereof.

8. R ａ and R ｂ are each methyl, the compound or a salt thereof according to claim 7.

9. R ４ The compound or a salt thereof according to any one of claims 1 to 8, wherein R is 5-(N,N-dimethylamino)penta-1-yl.

10. The following formula: [Chemical 2] The compound or a salt thereof according to claim 1, 7 or 8, which is a compound of the formula or a salt thereof.

11. Lipid particles comprising the compound or a salt thereof according to any one of claims 1 to 10.

12. The lipid particles according to claim 11, further comprising a non-cationic lipid.

13. The non-cationic lipid comprises cholesterol or a cholesterol derivative, a phospholipid, or a mixture of a phospholipid and cholesterol, and the cholesterol derivative is selected from cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof. The lipid particles according to claim 12.

14. The lipid particles according to claim 13, wherein the non-cationic lipid comprises a phospholipid, and the phospholipid is distearoyl phosphatidylcholine (DSPC).

15. The lipid particles according to any one of claims 11 to 14, further comprising a conjugate lipid that suppresses aggregation of the particles.

16. The lipid particles according to claim 15, wherein the conjugate lipid is a polyethylene glycol (PEG)-lipid conjugate.

17. The lipid particles according to claim 16, wherein the PEG-lipid conjugate is a PEG-dimyristyloxypropyl (PEG-DMA) conjugate.

18. The lipid particles according to any one of claims 11 to 17, further comprising a therapeutic agent.

19. The lipid particles according to claim 18, wherein the therapeutic agent is a nucleic acid therapeutic agent.

20. The lipid particles according to claim 19, wherein the nucleic acid therapeutic agent is an interfering RNA agent or mRNA.

21. The lipid particles according to claim 20, wherein the nucleic acid therapeutic agent is siRNA.

22. The lipid particles according to any one of claims 19 to 21, wherein the nucleic acid therapeutic agent comprises at least one modified nucleotide.

23. The lipid particles according to claim 22, wherein the nucleic acid therapeutic agent comprises at least one 2'-O-methyl (2'OMe) nucleotide.

24. A composition comprising the compound according to any one of claims 1 to 10 or a salt thereof.

25. A pharmaceutical composition comprising the lipid particles according to any one of claims 11 to 23 and a pharmaceutically acceptable carrier.

26. The lipid particles according to any one of claims 11 to 23 for use in the in vivo delivery of a therapeutic agent to a mammal.

27. Use of the lipid particles according to any one of claims 11 to 23 for preparing a medicament for the in vivo delivery of a therapeutic agent to a mammal.

28. A composition for treating a disease or disorder in a mammalian subject in need thereof, the composition comprising the lipid particles according to any one of claims 11 to 23.

29. The composition according to claim 28, wherein the disease or disorder is liver fibrosis, non-alcoholic steatohepatitis (NASH), or alcoholic steatohepatitis (ASH).

30. The composition according to claim 28, wherein the disease or disorder is non-alcoholic steatohepatitis (NASH) or alcoholic steatohepatitis (ASH) associated with liver fibrosis.

31. A composition for delivering a therapeutic agent to hepatic stellate cells (HSCs) in vivo or in vitro, the composition comprising the lipid particles according to any one of claims 11 to 23 and contacting the lipid particles with HSCs.

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