Cationic lipids containing silicon

Abstract

To provide cationic lipids that can be prepared using less expensive and more efficient processes.SOLUTION: The invention provides: cationic lipids of formula (I) in the figure, where R1, R2, R3 and R4 are defined as described herein; and methods of making these lipids. The invention also provides nucleic acid-lipid particles comprising the cationic lipids of formula (I), methods of making the lipid particles, and methods of delivering and / or administering the lipid particles.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Patent Application No. 62 / 758,108, filed November 9, 2018, which is incorporated herein by reference. [Background technology]

[0002] Nanoparticles containing cationic lipids have been used to deliver various active and therapeutic agents.Currently, the difficulty and cost associated with the preparation of the cationic lipid components of these nanoparticles limits their attractiveness for commercial development as delivery vehicles.Therefore, there is a need for additional cationic lipids that can be incorporated into lipid nanoparticles.In particular, there is a need for cationic lipids that can be prepared using cheaper and more efficient processes.There is also a need for cationic lipids that are relatively biodegradable, so that the associated lipid nanoparticles can be well degraded after delivery of active or therapeutic agents. Summary of the Invention

[0003] The present invention provides cationic lipid that can be prepared using cheaper and more efficient process.Furthermore, when this cationic lipid is incorporated into lipid nanoparticle, it is effective for delivering active or therapeutic agent such as nucleic acid.This cationic lipid is also biodegradable, so that the associated lipid nanoparticle can be decomposed well after delivery of active or therapeutic agent.

[0004] In one embodiment, the present invention provides a method for the preparation of a compound of formula (I): [ka] (In the formula, R 1 is C2~C 30 is a hydrocarbyl; R 2 is C2~C 30 is a hydrocarbyl; R 3 is C2~C 30is a hydrocarbyl; X is a divalent C2-C8 alkyl; R 4 is NR a R b and; R a and R b are each independently selected from the group consisting of methyl, ethyl, propyl, cyclopropyl, and butyl, wherein methyl, ethyl, propyl, cyclopropyl, and butyl are optionally substituted with hydroxy; or R a and R b together with the nitrogen to which they are attached form an aziridine ring, azetidine ring, proline ring, piperidine ring, piperazine ring, or morpholine ring, which ring is optionally substituted with hydroxyl or C1-C6 alkyl optionally substituted with hydroxyl).

[0005] The present invention also provides compositions comprising 1) a compound of formula (I) and 2) an active or therapeutic agent.

[0006] The present invention also provides lipid nanoparticles comprising a compound of formula (I).

[0007] The present invention also provides lipid nanoparticles comprising (a) one or more active or therapeutic agents, (b) a compound of formula (I), (c) a non-cationic lipid, and (d) a conjugated lipid. Typically, the one or more agents are encapsulated within the lipid nanoparticle.

[0008] The present invention also provides pharmaceutical compositions comprising 1) a compound of formula (I) and 2) one or more active or therapeutic agents.

[0009] The present invention also provides a pharmaceutical composition comprising the lipid nanoparticles of the present invention and a pharma- ceutically acceptable carrier.

[0010] The invention also provides a method for delivering an active or therapeutic agent (eg, a nucleic acid) to an animal, comprising administering to the animal a nanoparticle of the invention.

[0011] The present invention also provides methods for introducing a nucleic acid into a cell, comprising contacting the cell in vivo, ex vivo, or in vitro with a nucleic acid-lipid particle described herein.

[0012] The present invention also provides a method for treating a disease or disorder in an animal, comprising administering to the animal a therapeutically effective amount of a nucleic acid-lipid particle described herein.

[0013] The invention also provides processes and intermediates disclosed herein that are useful for making the compounds, formulations, or nanoparticles of the invention. For example, in one embodiment, the invention provides a compound of formula (I): [ka] (In the formula, R 4 is NR as described herein. a R b which comprises preparing a corresponding compound of formula (Ia): [ka] (In the formula, R 4a is bromo or chloro) to the formula HNR a R b with an amine to obtain a compound of formula (I). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] definition As used herein, the following terms have the meanings ascribed to them unless stated otherwise.

[0015] The term "interfering RNA" or "RNAi" or "interfering RNA sequence" refers to a 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 the 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, an interfering RNA refers to a single-stranded RNA that is complementary to a target mRNA sequence, or a double-stranded RNA formed by two complementary strands or by one self-complementary strand. An interfering RNA may have substantial or complete identity to a target gene or sequence, or may contain a mismatch region (i.e., a mismatch motif). The sequence of an interfering RNA may correspond to a full-length target gene or a subsequence thereof.

[0016] Interfering RNA includes "small interfering RNA" or "siRNA", e.g., an interfering RNA that is about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary strand of a double-stranded siRNA). The sequences are 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably about 18-22, 19-20, or 19-21 base pairs in length.) 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 siRNA include, but are not limited to, 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 single-stranded molecules, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; double-stranded polynucleotide molecules having a hairpin secondary structure with self-complementary sense and antisense regions; and circular single-stranded polynucleotide molecules having a stem with two or more loop structures and self-complementary sense and antisense regions, where the circular polynucleotide can be processed in vivo or in vitro to produce an active double-stranded siRNA molecule.

[0017] Preferably, the siRNA is chemically synthesized. siRNA can also be produced by cleaving longer dsRNA (e.g., dsRNA greater than about 25 nucleotides in length) with E. coli RNase III or Dicer. These enzymes process dsRNA into biologically active siRNAs (see, e.g., Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al., Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); and Robertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, the dsRNA is at least 50 nucleotides long to about 100, 200, 300, 400 or 500 nucleotides long. The dsRNA can be 1000, 1500, 2000, 5000 nucleotides long 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 duplex with a hairpin loop).

[0018] As used herein, the term "mismatch motif" or "mismatch region" refers to a portion of an interfering RNA (e.g., siRNA, aiRNA, miRNA) sequence that does not have 100% complementarity to its target sequence. An interfering RNA may have at least one, two, three, four, five, six, or more mismatch regions. The mismatch regions may be contiguous or separated by one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more nucleotides. A mismatch motif or mismatch region may comprise one nucleotide, or may comprise two, three, four, five, or more nucleotides.

[0019] An "effective amount" or "therapeutically effective amount" of an active or therapeutic agent, such as a nucleic acid (e.g., an interfering RNA or mRNA), is an amount sufficient to produce a desired effect, such as inhibition of expression of a target sequence compared to the normal expression level detected in the absence of an interfering RNA, or mRNA-specific expression of the amount of protein that is expressed in vivo and produces a desired biological effect therein. Inhibition of expression of a target gene or target sequence is achieved when the value obtained using an 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 an active form of a protein normally expressed in a cell type in the body, and a therapeutically effective amount of mRNA is an amount that produces an amount of the encoded protein that is at least 50% (e.g., at least 60%, or at least 70%, or at least 80%, or at least 90%) of the amount of protein normally expressed in that cell type in a healthy individual. Suitable assays for measuring expression of target genes or target sequences include, for example, examination of protein or RNA levels using techniques known to those of skill in the art, such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those of skill in the art.

[0020] By "reducing", "reducing", "reducing" or "reducing" an immune response by an interfering RNA is intended to mean a detectable decrease in the immune response to a given interfering RNA (e.g., a modified interfering RNA). The amount of decrease in the immune response by a modified interfering RNA can be measured relative to the level of the immune response in the presence of an unmodified interfering RNA. A 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 an unmodified interfering RNA. A decrease in the immune response to an interfering RNA is usually measured by a decrease in cytokine production (e.g., IFNγ, IFNα, TNFα, IL-6 or IL-12) by responder cells in vitro or a decrease in cytokine production in the serum of a mammalian subject after administration of an interfering RNA.

[0021] By "reducing", "reducing", "reducing" or "reducing" an immune response to an mRNA is intended to mean a detectable decrease in the immune response to a given mRNA (e.g., a modified mRNA). The amount of decrease in the immune response to a modified mRNA can be measured relative to the level of the immune response in the presence of an unmodified mRNA. A 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 an unmodified mRNA. A decrease in the immune response to an mRNA is usually measured by a decrease in cytokine production (e.g., IFNγ, IFNα, TNFα, IL-6 or IL-12) by responder cells in vitro or in the serum of a mammalian subject following administration of the mRNA.

[0022] As used herein, the term "responder cell" refers to a cell, preferably a mammalian cell, that produces a detectable immune response when contacted with an immunostimulatory interfering RNA, such as an unmodified siRNA. Exemplary responder cells include, for example, dendritic cells, macrophages, peripheral blood mononuclear cells (PBMCs), splenocytes, and the like. The detectable immune response includes the production of cytokines or growth factors, such as, for example, 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.

[0023] "Substantial identity" refers to a sequence that hybridizes to a reference sequence under stringent conditions or has a particular percent identity over a particular region of the reference sequence.

[0024] 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 to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize, particularly at higher temperatures. An extensive guide to nucleic acid hybridization is 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). In general, stringent conditions are determined by the thermal melting point (T) of a particular sequence at a defined ionic strength pH. m ) is chosen to be about 5 to 10°C lower than T mis the temperature (at defined ionic strength, pH, and nuclear concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (if the target sequence is present in excess, T m (50% of the probes are occupied at equilibrium at 100°C). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization.

[0025] Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5xSSC, and 1% SDS, incubated at 42°C, or 5xSSC, 1% SDS, incubated at 65°C, washed in 0.2xSSC, and 0.1% SDS at 65°C. For PCR, low stringency amplification is usually at a temperature of about 36°C, while annealing temperatures can vary from about 32°C to 48°C depending on primer length. For high stringency PCR amplification, temperatures are usually about 62°C, while high stringency annealing temperatures can range from about 50°C to about 65°C depending on primer length and specificity. Typical cycling conditions for both high and low stringency amplification include a denaturation step at 90°C to 95°C for 30 seconds to 2 minutes, an annealing step lasting 30 seconds to 2 minutes, and an extension step at about 72°C for 1 to 2 minutes. Protocols and guidelines for low stringency and high stringency amplification reactions are provided, for example, in Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. NY (1990).

[0026] 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 copies of nucleic acids are generated using the maximum codon degeneracy permitted by the genetic code. In such cases, nucleic acids usually hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include hybridization in a buffer of 40% formamide, 1M NaCl, 1% SDS at 37°C, and washing in 1xSSC at 45°C. Positive hybridization is at least twice background. Those skilled in the art will readily understand that alternative hybridization and washing conditions can be utilized to obtain similar stringency conditions. Further guidelines for determining hybridization parameters are provided in numerous references, for example, Current Protocols in Molecular Biology, Ausubel et al., eds.

[0027] The term "substantially identical" or "substantial identity" in the context of two or more nucleic acids refers to two or more sequences or subsequences that are the same or have a certain percentage of identical nucleotides (i.e., at least about 60%, preferably at least about 65%, 70%, 75%, 80%, 85%, 90% or 95% identity over a particular region) when compared and aligned for maximum correspondence over a comparison window or over a designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of a sequence as well, where the context indicates. Preferably, the substantial identity exists over a region that is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

[0028] For sequence comparison, one sequence usually serves as a reference sequence to compare with test sequence.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, partial sequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence to reference sequence based on program parameters.

[0029] As used herein, a "comparison window" includes reference to any one section of several contiguous positions selected from the group consisting of about 5 to about 60, typically about 10 to about 45, and more typically about 15 to about 30, that can be compared between a sequence and a reference sequence of the same number of contiguous positions after optimal alignment of the two sequences. Methods of sequence alignment for comparison are well known in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970), the search for similarity 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, for example, Current Protocols in Molecular Biology, Ausubel et al., eds. (1995 supplement)).

[0030] 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 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 analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0031] BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarity provided by 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, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability in the comparison between test nucleic acid and reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0032] The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides, in either single-stranded or double-stranded form, and includes DNA and RNA. DNA can be in the form of, for example, antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations 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 synthetic, natural, and non-natural nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties as the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless expressly limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as 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 sequence specified. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is replaced with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups."Base" includes purines and pyrimidines, which further include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0033] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises a partial or full-length coding sequence necessary for the production of a polypeptide or a precursor polypeptide.

[0034] As used herein, "gene product" refers to the product of a gene, such as an RNA transcript or a polypeptide.

[0035] The term "lipids" refers to a group of organic compounds, including but not limited to esters of fatty acids, that are insoluble in water but soluble in many organic solvents. They are generally divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes, (2) "complex lipids," which include phospholipids and glycolipids, and (3) "derived lipids," such as steroids.

[0036] 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 refer to particles made of lipids (e.g., cationic lipids, non-cationic lipids, and conjugated lipids that prevent particle aggregation) and nucleic acids, in which the nucleic acid (e.g., siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, mRNA, self-amplifying RNA, or plasmid (including interfering RNA or a plasmid from which mRNA is transcribed)) is encapsulated within the lipid. In one embodiment, the nucleic acid is at least 50% encapsulated within the lipid, in one embodiment, the nucleic acid is at least 75% encapsulated within the lipid, in one embodiment, the nucleic acid is at least 90% encapsulated within the lipid, and in one embodiment, the nucleic acid is completely encapsulated within the lipid. LNPs typically contain cationic lipids, non-cationic lipids, and lipid conjugates (e.g., PEG-lipid conjugates). LNPs are extremely useful for systemic applications because they can exhibit extended circulatory lifetimes following intravenous (iv) injection, can accumulate at distal sites (e.g., sites physically separated from the site of administration), and can mediate expression of transfected genes or silencing of target gene expression at these distal sites.

[0037] The lipid particles (e.g., LNPs) of the present invention typically have an average diameter of about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm, and are substantially non-toxic. Furthermore, the nucleic acid, when present in the lipid particles of the present invention, is resistant to degradation by nucleases 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 incorporated by reference herein in their entireties for all purposes.

[0038] As used herein, "lipid-encapsulated" can refer to lipid particles that provide an active or therapeutic agent, such as a nucleic acid (e.g., an interfering RNA or an mRNA), that is fully encapsulated, partially encapsulated, or both. In preferred embodiments, the nucleic acid is fully encapsulated in the lipid particle (e.g., to form an SPLP, pSPLP, LNP, or other nucleic acid-lipid particle).

[0039] The term "lipid conjugate" refers to a conjugated lipid that inhibits lipid particle aggregation. 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. Pat. No. 5,885,613, which is incorporated by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG can be directly conjugated to lipid or linked to lipid via a linker moiety. For example, any linker moiety suitable for coupling PEG to lipid can be used, including ester-free linker moieties and ester-containing linker moieties. In a preferred embodiment, ester-free linker moieties are used.

[0040] The term "amphipathic lipid" refers, in part, to any suitable material in which the hydrophobic portion of the lipid material orients toward the hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. The hydrophilic character comes from the presence of polar or charged groups, such as carbohydrate, phosphate, carboxyl, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other similar groups. Hydrophobicity can be imparted by the inclusion of non-polar groups, including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic group(s), alicyclic group(s), or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0041] 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 that lack phosphorus, such as sphingolipids, glycosphingolipid families, diacylglycerols, and β-acyloxyacids, are also included in the group designated as amphipathic lipids.Furthermore, the above-mentioned amphipathic lipids can be mixed with other lipids, including triglycerides and sterols.

[0042] The term "neutral lipid" refers to any of several lipid species that exist in either uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0043] The term "non-cationic lipid" refers to any amphipathic lipid and any other neutral or anionic lipid.

[0044] 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.

[0045] The term "cationic lipid" refers to a compound of formula (I) as described herein.

[0046] The term "hydrophobic lipid" refers to compounds that have apolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups are optionally substituted with one or more aromatic group(s), alicyclic group(s), or heterocyclic group(s).Suitable examples include but are not limited to diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0047] The term "fusogenic" refers to the ability of a lipid particle, such as an LNP, to fuse with the membrane of a cell. The membrane can be either the plasma membrane or a membrane surrounding an organelle, such as an endosome, nucleus, etc.

[0048] As used herein, the term "aqueous solution" refers to a composition that comprises, in whole or in part, water.

[0049] As used herein, the term "organic lipid solution" refers to a composition that comprises, in whole or in part, an organic solvent with a lipid.

[0050] As used herein, a "distant site" refers to a physically separated site, including but not limited to adjacent capillary beds, and including sites distributed generally throughout the organism.

[0051] "Serum stability" in the context of 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.

[0052] As used herein, "systemic delivery" refers to delivery of lipid particles that results in widespread biodistribution of active or therapeutic agents, such as interfering RNA or mRNA, in vivo. Some administration techniques can result in systemic delivery of a particular agent, while others do not. Systemic delivery means that a useful amount, preferably a therapeutic amount, of the agent is exposed to most of the body. To obtain widespread biodistribution, a blood lifetime is generally required such that the agent does not undergo rapid degradation or clearance (such as by first-pass organs (liver, lung, etc.) or rapid non-specific cell binding) before reaching disease sites distal to the administration site. 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.

[0053] As used herein, "local delivery" refers to directly delivering active or therapeutic agents, such as interference RNA or mRNA, to target sites in vivo.For example, drugs can be locally delivered by direct injection to disease sites such as tumors, or other target sites such as inflammation sites, or target organs such as liver, heart, pancreas, kidney, etc.

[0054] The term "mammal" refers to any mammalian species, such as, for example, humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, farm animals, etc.

[0055] The term "cancer" refers to any member of a class of diseases characterized by uncontrolled proliferation of abnormal cells. The term includes all known cancers and tumor conditions, whether characterized as malignant, benign, soft tissue, or solid, as well as all stages and grades of cancer, including pre- and post-metastatic cancer. Examples of different types of cancer include, but are not limited to, lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, stomach (gastric) cancer, esophageal cancer; gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer, kidney cancer (e.g., renal cell carcinoma), cancer of the central nervous system, glioblastoma, skin cancer, lymphoma, choriocarcinoma, head and neck cancer, osteogenic sarcoma, and blood cancer. Non-limiting examples of specific types of liver cancer include hepatocellular carcinoma (HCC), secondary liver cancer (e.g., those resulting from metastasis of some other non-hepatic cancer cell type), and hepatoblastoma. As used herein, a "tumor" comprises one or more cancer cells.

[0056] The term "anion precursor group" includes groups capable of forming ions at physiological pH. For example, the term includes the groups -CO2H, -OP(=O)(OH), -OS(=O)2(OH), -OS(=O)(OH), and -B(OH). In one embodiment, the anion precursor is -CO2H.

[0057] In certain embodiments, PEG-C-DMA has the following structure: [ka] (wherein n is selected such that the resulting polymer chain has a molecular weight of about 1000 to about 3000). In another embodiment, n is selected such that the resulting polymer chain has a molecular weight of about 2000. PEG-C-DMA can be prepared as described in Heyes et al, Synthesis and Characterization of Novel Poly(Ethylene Glycol)-lipid Conjugates Suitable for use in Drug Delivery," Journal of Controlled Release, 2006, and U.S. Pat. No. 8,936,942.

[0058] Description of the embodiments The present invention provides novel serum-stable lipid particles comprising one or more active 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).

[0059] In one aspect, the invention provides lipid particles comprising: (a) one or more active or therapeutic agents; (b) one or more cationic lipids that constitute from about 30 mol% to about 85 mol% of the total lipid present in the particle; (c) one or more non-cationic lipids that constitute from about 13 mol% to about 49.5 mol% of the total lipid present in the particle; and (d) one or more conjugated lipids that inhibit particle aggregation, that constitute from about 0.1 mol% to about 10 mol% of the total lipid present in the particle.

[0060] In one aspect, the invention provides lipid particles comprising: (a) one or more active or therapeutic agents; (b) one or more cationic lipids that constitute from about 50 mol% to about 85 mol% of the total lipid present in the particle; (c) one or more non-cationic lipids that constitute from about 13 mol% to about 49.5 mol% of the total lipid present in the particle; and (d) one or more conjugated lipids that inhibit particle aggregation, that constitute from about 0.5 mol% to about 2 mol% of the total lipid present in the particle.

[0061] In certain embodiments, the active agent or therapeutic agent is fully encapsulated within the lipid portion of the lipid particle such that the active agent or therapeutic agent in the lipid particle is resistant to enzymatic degradation, e.g., by nucleases or proteases, in aqueous solution, hi certain other embodiments, the lipid particle is substantially non-toxic to mammals, such as humans.

[0062] In some embodiments, the active agent or therapeutic agent comprises a nucleic acid. In certain cases, the nucleic acid comprises an interfering RNA molecule, such as, for example, siRNA, aiRNA, miRNA, or a mixture thereof. In certain other cases, the nucleic acid comprises a single-stranded or double-stranded DNA, RNA, or a DNA / RNA hybrid, such as, for example, an antisense oligonucleotide, a ribozyme, a plasmid, an immunostimulatory oligonucleotide, or a mixture thereof. In certain cases, the nucleic acid comprises an mRNA molecule.

[0063] In other embodiments, the active or therapeutic agent comprises a peptide or polypeptide. In certain cases, the peptide or polypeptide comprises an antibody, such as, for example, a polyclonal antibody, a monoclonal antibody, an antibody fragment; a humanized antibody, a recombinant antibody, a recombinant human antibody, a Primatized™ antibody, or a mixture thereof. In certain other cases, the peptide or polypeptide comprises a cytokine, a growth factor, an apoptotic factor, a differentiation inducer, a cell surface receptor, a ligand, a hormone, a small molecule (e.g., a small organic molecule or chemical compound), or a mixture thereof.

[0064] In one embodiment, the active or therapeutic agent comprises an siRNA. In one embodiment, the siRNA molecule comprises a double-stranded region of about 15 to about 60 nucleotides in length (e.g., about 15-60, 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 molecule of the present invention can silence expression of a target sequence in vitro and / or in vivo.

[0065] In some embodiments, the siRNA molecule comprises at least one modified nucleotide. In certain preferred embodiments, the siRNA molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides in the double-stranded region. In certain cases, the siRNA comprises 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 preferred embodiments, less than about 25% (e.g., less than about 25%, less than 20%, less than 15%, less than 10%, or less than 5%) or about 1% to about 25% (e.g., about 1% to 25%, 5% to 25%, 10% to 25%, 15% to 25%, 20% to 25%, or 10% to 20%) of the nucleotides in the double-stranded region comprise modified nucleotides.

[0066] 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'-deoxy nucleotides, 2'-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof. In preferred embodiments, 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.

[0067] siRNA may contain modified nucleotides in one strand (i.e., sense or antisense) or both strands of the double-stranded region of the siRNA molecule. Preferably, uridine and / or guanosine nucleotides are modified at selective positions in the double-stranded region of the siRNA duplex. With respect to uridine nucleotide modifications, at least one, two, three, four, five, six, or more of the uridine nucleotides in the sense strand and / or antisense strand may be modified uridine nucleotides, such as 2'OMe-uridine nucleotides. In some embodiments, all of the uridine nucleotides in the sense strand and / or antisense strand are 2'OMe-uridine nucleotides. With respect to guanosine modifications, at least one, two, three, four, five, six, or more of the guanosine nucleotides in the sense strand and / or antisense strand may be modified guanosine nucleotides, such as 2'OMe-guanosine nucleotides. In some embodiments, all of the guanosine nucleotides in the sense strand and / or antisense strand are 2'OMe-guanosine nucleotides.

[0068] In certain embodiments, at least one, two, three, four, five, six, seven, or more 5'-GU-3' motifs of the siRNA sequence may be modified, for example, by introducing mismatches to remove the 5'-GU-3' motifs and / or by introducing modified nucleotides, such as 2'OMe nucleotides. The 5'-GU-3' motifs may be present in the sense strand, the antisense strand, or both strands of the siRNA sequence. The 5'-GU-3' motifs may be adjacent to each other or, alternatively, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides.

[0069] 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 advantageously 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 into the siRNA sequence, for example, into 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 one of skill in the art that the immune stimulatory properties of modified and corresponding unmodified siRNA molecules can be determined, for example, by measuring INF-α and / or IL-6 levels about 2 to about 12 hours after systemic administration in a mammal or transfection of mammalian responder cells using a suitable lipid-based delivery system (such as the LNP delivery system disclosed herein).

[0070] In certain embodiments, modified siRNA molecules have an IC value less than or equal to the IC of the corresponding unmodified siRNA. 50 (i.e., IC50 less than 10-fold higher than the half-maximal inhibitory concentration) 50 (i.e., the modified siRNA has an IC 50 IC less than 10 times 50 In other embodiments, the modified siRNA has the IC 50 IC less than 3 times 50 In yet other embodiments, the modified siRNA has an IC 50 IC less than twice the 50 Using methods well known to those of skill in the art, dose-response curves can be generated and the IC of the modified siRNA and the corresponding unmodified siRNA can be calculated.50 It will be readily apparent to one of skill in the art that these values ​​can be readily determined.

[0071] In yet another embodiment, the modified siRNA molecule is capable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the expression of the target sequence compared to the corresponding unmodified siRNA.

[0072] In some embodiments, the siRNA molecule does not contain a phosphate backbone modification, for example in the sense strand and / or 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 antisense strand of the double-stranded region. In a preferred embodiment, the siRNA does not contain a phosphate backbone modification.

[0073] In further embodiments, the siRNA does not contain 2'-deoxynucleotides, for example in the sense strand and / or antisense strand of the double-stranded region. In yet further embodiments, the siRNA contains one, two, three, four or more 2'-deoxynucleotides, for example in the sense strand and / or antisense strand of the double-stranded region. In preferred embodiments, the siRNA does not contain 2'-deoxynucleotides.

[0074] In certain cases, the nucleotide at the 3' end of the double-stranded region of the sense strand and / or antisense strand is not a modified nucleotide. In certain other cases, the nucleotide near the 3' end of the double-stranded region of the sense strand and / or antisense strand (e.g., within 1, 2, 3, or 4 nucleotides of the 3' end) is not a modified nucleotide.

[0075] The siRNA molecules described herein may have a 3' overhang of one, two, three, four or more nucleotides on one or both sides of the double-stranded region, or may lack an overhang (i.e., have a blunt end) on one or both sides of the double-stranded region. Preferably, the siRNA has a 3' overhang 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 comprises one, two, three, four or more nucleotides, such as 2'-deoxy (2'H) nucleotides. In certain preferred embodiments, the 3' overhang comprises 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 comprise modified nucleotides.Non-limiting examples of modified nucleotides are described above and include 2'OMe nucleotides, 2'-deoxy-2'F nucleotides, 2'-deoxy nucleotides, 2'-O-2-MOE nucleotides, LNA nucleotides, and mixtures thereof.In preferred embodiments, one, two, three, four, or more nucleotides in the 3' overhang present on the sense strand and / or antisense strand of siRNA comprise 2'OMe nucleotides (e.g., 2'OMe purine and / or pyrimidine nucleotides), such as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, 2'OMe-cytosine nucleotides, and mixtures thereof.

[0076] The siRNA may comprise at least one or a cocktail of unmodified and / or modified siRNA sequences (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) that silence the expression of a target gene. The cocktail of siRNAs may comprise sequences that target the same region or domain (e.g., "hotspot") 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 two, three, four, five, six, seven, eight, nine, ten, 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 two, three, four, five, six, seven, eight, nine, ten, or more) target genes are present in the cocktail.

[0077] 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 part 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 part 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 part thereof.

[0078] 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.

[0079] In the lipid nanoparticles of the present invention, the cationic lipid may be selected from the compounds of formula (I) described herein.

[0080] In some embodiments, the cationic lipid may constitute about 30 mol% to about 90 mol%, about 30 mol% to about 85 mol%, about 30 mol% to about 80 mol%, about 30 mol% to about 75 mol%, about 30 mol% to about 70 mol%, about 30 mol% to about 65 mol%, or about 30 mol% to about 60 mol% of the total lipid present in the particle.

[0081] In some embodiments, the cationic lipid may constitute about 40 mol% to about 90 mol%, about 40 mol% to about 85 mol%, about 40 mol% to about 80 mol%, about 40 mol% to about 75 mol%, about 40 mol% to about 70 mol%, about 40 mol% to about 65 mol%, or about 40 mol% to about 60 mol% of the total lipid present in the particle.

[0082] 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 lipid present in the particle.

[0083] 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 lipid present in the particle.

[0084] In still 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 lipid present in the particle.

[0085] In further embodiments, the cationic lipid may constitute about 70 mol% to about 90 mol%, about 70 mol% to about 85 mol%, about 70 mol% to about 80 mol%, about 75 mol% to about 90 mol%, about 75 mol% to about 85 mol%, or about 80 mol% to about 90 mol% of the total lipid present in the particle.

[0086] In additional embodiments, the cationic lipid may constitute (at least) about 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 mol % (or any fraction or range therein) of the total lipid present in the particle.

[0087] In the lipid particles of the present invention, the non-cationic lipids may, for example, comprise one or more anionic lipids and / or neutral lipids. In a preferred embodiment, the non-cationic lipids comprise 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.

[0088] 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.

[0089] The phospholipid may be a neutral lipid, including, but not limited to, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), and mixtures thereof. In certain preferred embodiments, the phospholipid is DPPC, DSPC, or a mixture thereof.

[0090] In some embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) may comprise 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 lipid present in the particle. When the non-cationic lipid is a mixture of phospholipid and cholesterol or a cholesterol derivative, the mixture may comprise up to about 40, 50, or 60 mol% of the total lipid present in the particle.

[0091] In other embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) may constitute from about 10 mol% to about 49.5 mol%, about 13 mol% to about 49.5 mol%, about 15 mol% to about 49.5 mol%, about 20 mol% to about 49.5 mol%, about 25 mol% to about 49.5 mol%, about 30 mol% to about 49.5 mol%, about 35 mol% to about 49.5 mol%, or about 40 mol% to about 49.5 mol% of the total lipid present in the particle.

[0092] In still other embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) may constitute from about 10 mol% to about 45 mol%, about 13 mol% to about 45 mol%, about 15 mol% to about 45 mol%, about 20 mol% to about 45 mol%, about 25 mol% to about 45 mol%, about 30 mol% to about 45 mol%, or about 35 mol% to about 45 mol% of the total lipid present in the particle.

[0093] In still other embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) may constitute from about 10 mol% to about 40 mol%, about 13 mol% to about 40 mol%, about 15 mol% to about 40 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 40 mol%, or about 30 mol% to about 40 mol% of the total lipid present in the particle.

[0094] In further embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) may constitute from about 10 mol% to about 35 mol%, about 13 mol% to about 35 mol%, about 15 mol% to about 35 mol%, about 20 mol% to about 35 mol%, or about 25 mol% to about 35 mol% of the total lipid present in the particle.

[0095] In still further embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) may constitute about 10 mol% to about 30 mol%, about 13 mol% to about 30 mol%, about 15 mol% to about 30 mol%, about 20 mol% to about 30 mol%, about 10 mol% to about 25 mol%, about 13 mol% to about 25 mol%, or about 15 mol% to about 25 mol% of the total lipid present in the particle.

[0096] In additional embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) may constitute (at least) about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol % (or any fraction or range therein) of the total lipid present in the particle.

[0097] In certain preferred embodiments, the non-cationic lipid comprises about 31.5 mol% to about 42.5 mol% cholesterol or a derivative thereof of the total lipid present in the particle. As a non-limiting example, a phospholipid-free lipid particle of the present invention may comprise about 37 mol% cholesterol or a derivative thereof of the total lipid present in the particle. In other preferred embodiments, the phospholipid-free lipid particles of the present invention may comprise about 30 mol% to about 45 mol%, about 30 mol% to about 40 mol%, about 30 mol% to about 35 mol%, about 35 mol% to about 45 mol%, about 40 mol% to about 45 mol%, about 32 mol% to about 45 mol%, about 32 mol% to about 42 mol%, about 32 mol% to about 40 mol%, about 34 mol% to about 45 mol%, about 34 mol% to about 42 mol%, about 34 mol% to about 40 mol%, or about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 mol% (or any fraction thereof or range therein) of cholesterol or a derivative thereof of the total lipid present in the particle.

[0098] In certain other preferred embodiments, the non-cationic lipid comprises a mixture of (i) phospholipids 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, a lipid particle comprising a mixture of phospholipids and cholesterol may 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 (i) about 3 mol% to about 15 mol%, about 4 mol% to about 15 mol%, about 4 mol% to about 12 mol%, about 4 mol% to about 10 mol%, about 4 mol% to about 8 mol%, about 5 mol% to about 12 mol%, about 5 mol% to about 9 mol%, about 6 mol% to about 12 mol%, about 6 mol% to about 10 mol%, or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% (or any fraction or range therein) of phospholipids of the total lipid present in the particle; and (ii) about 25 mol% to about 45 mol%, about 30 mol% to about 45 mol% of the total lipid present in the particle. 1%, 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 thereof or range therein) of cholesterol or a derivative thereof.

[0099] In a further preferred embodiment, the non-cationic lipid comprises a mixture of (i) about 10 mol% to about 30 mol% of a phospholipid based on the total lipid present in the particle, and (ii) about 10 mol% to about 30 mol% of cholesterol or a derivative thereof based on the total lipid present in the particle. As a non-limiting example, a lipid particle comprising a mixture of phospholipid and cholesterol may comprise about 20 mol% DPPC and about 20 mol% cholesterol based on the total lipid present in the particle. 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 thereof or range therein) of phospholipids of the total lipid present in the particle; (ii) A mixture of about 10 mol% to about 30 mol%, about 10 mol% to about 25 mol%, about 10 mol% to about 20 mol%, about 15 mol% to about 30 mol%, about 20 mol% to about 30 mol%, about 15 mol% to about 25 mol%, about 12 mol% to about 28 mol%, about 14 mol% to about 26 mol%, or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mol% (or any fraction thereof or range therein) of the total lipid present in the particle with cholesterol or a derivative thereof.

[0100] Conjugated lipids In the lipid particles of the present invention (e.g., LNPs (e.g., containing an interfering RNA such as siRNA, or mRNA)), the conjugated lipid may comprise, for example, one or more of the following: polyethylene glycol (PEG)-lipid conjugates, polyamide (ATTA)-lipid conjugates, or mixtures thereof. In a preferred embodiment, the nucleic acid-lipid particle comprises either a PEG-lipid conjugate or an ATTA-lipid conjugate. The conjugated lipid may comprise, for example, a PEG-lipid, including PEG-diacylglycerol (DAG), PEG dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmityloxypropyl (C16), PEG-distearyloxypropyl (C18), or mixtures thereof.

[0101] 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 December 31, 2008, the disclosure of which is incorporated herein by reference in its entirety for all purposes.Still 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-w-methyl-poly(ethylene glycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in US Pat. No. 7,404,969, the disclosure of which is incorporated by reference herein in its entirety for all purposes.

[0102] The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from 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 preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0103] In certain cases, the conjugated lipid (e.g., a PEG-lipid conjugate) may constitute from about 0.1 to about 10% (or any fraction or range therein) of the total lipid present in the particle. In certain cases, the conjugated lipid (e.g., a PEG-lipid conjugate) may 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 thereof or range therein) of the total lipid present in the particle.

[0104] In the lipid particles of the present invention, active agent or therapeutic agent is completely encapsulated in the lipid portion of the particle, which can protect the active agent or therapeutic agent from nuclease degradation.In a preferred embodiment, the LNPs containing nucleic acid such as interfering RNA (e.g., siRNA) or mRNA are completely encapsulated in the lipid portion of the particle, which can protect the nucleic acid from nuclease degradation.In certain cases, the nucleic acid in the LNPs is not substantially degraded after the particle is exposed to nuclease at 37°C for at least about 20, 30, 45, or 60 minutes.In certain other cases, the nucleic acid in the LNPs is not substantially degraded after the particle is incubated in serum at 37°C for at least about 30, 45, or 60 minutes or 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 an siRNA) is complexed with the lipid portion of the particle.One advantage of the formulations of the present invention is that the lipid particle compositions are substantially non-toxic to mammals, such as humans.

[0105] The term "fully encapsulated" indicates that the active or therapeutic agent in the lipid particles is not significantly degraded after exposure to serum or nuclease or protease assays that can significantly degrade free DNA, RNA or protein. In a fully encapsulated system, preferably less than about 25% of the active or therapeutic agent in the particles is degraded, more preferably less than about 10%, and most preferably less than about 5% of the active or therapeutic agent in the particles is degraded in a process that would normally degrade 100% of the free active or therapeutic agent. In the context of nucleic acid therapeutics, complete encapsulation can be determined by Oligreen® assay. Oligreen® is an ultrasensitive fluorescent nucleic acid stain for quantifying oligonucleotides and single-stranded DNA or RNA in solution (available from Invitrogen Corporation; Carlsbad, Calif.). "Completely encapsulated" also indicates that the lipid particles are serum stable, i.e., they are not rapidly degraded into their components upon in vivo administration.

[0106] In another aspect, the present invention provides a lipid particle (e.g., LNP) composition comprising a plurality of lipid particles. In a preferred embodiment, an active agent or therapeutic agent (e.g., nucleic acid) comprises 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%, or about 50% to about 95% of the lipid particle (e.g., LNP). %, 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 thereof or range therein) of the active agent or therapeutic agent is completely encapsulated within the lipid portion of the lipid particle (e.g., LNP) such that about

[0107] Typically, lipid particles (e.g., LNPs) of the invention have a lipid:active agent (e.g., lipid:nucleic acid) ratio (mass / mass ratio) of about 1 to about 100. In some cases, the lipid:active 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 preferred embodiments, lipid particles of the invention have a lipid:active agent (e.g., lipid:nucleic acid) ratio (mass / mass ratio) of about 5 to about 15, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (or any fraction or range therein).

[0108] Typically, the lipid particles (eg, LNPs) of the present invention have an average diameter of about 40 nm to about 150 nm. In preferred embodiments, lipid particles (e.g., LNPs) of the present invention have an average diameter of about 40 nm to about 130 nm, about 40 nm to about 120 nm, about 40 nm to about 100 nm, about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 120 nm, about 60 nm to about 110 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 70 nm to about 120 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, or less than about 120 nm, less than about 110 nm, less than about 100 nm, less than about 90 nm, or less than about 80 nm (or any fraction thereof or range therein).

[0109] In one particular embodiment of the invention, the LNP comprises (a) one or more unmodified and / or modified nucleic acid molecules (e.g., siRNA, aiRNA, interfering RNA that silences expression of a target gene, such as miRNA, or mRNA that causes expression of a target protein), (b) a cationic lipid that constitutes about 56.5 mol% to about 66.5 mol% of the total lipid present in the particle, (c) a non-cationic lipid that constitutes about 31.5 mol% to about 42.5 mol% of the total lipid present in the particle, and (d) a conjugated lipid that inhibits particle aggregation, that constitutes about 1 mol% to about 2 mol% of the total lipid present in the particle. This particular embodiment of the LNP is generally referred to herein as a "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 conjugated lipid is a PEG-DAA conjugate. While these are preferred embodiments of the 1:62 formulation, one of skill in the art will understand that other cationic lipids, non-cationic lipids (including other cholesterol derivatives), and conjugated lipids can be used in the 1:62 formulations described herein.

[0110] In another particular embodiment of the invention, the LNP comprises (a) one or more unmodified and / or modified nucleic acid molecules (e.g., an interfering RNA that silences expression of a target gene, such as an siRNA, an aiRNA, an miRNA, or an mRNA that causes expression of a target protein), (b) a cationic lipid that constitutes about 52 mol% to about 62 mol% of the total lipid present in the particle, (c) a non-cationic lipid that constitutes about 36 mol% to about 47 mol% of the total lipid present in the particle, and (d) a conjugated lipid that inhibits particle aggregation, that constitutes about 1 mol% to about 2 mol% of the total lipid present in the particle. This particular embodiment of the LNP is generally referred to herein as a "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 phospholipid (such as DPPC) and cholesterol (wherein the phospholipid constitutes about 5 mol% to about 9 mol% (e.g., about 7.1 mol%) of the total lipid present in the particle, and the cholesterol (or cholesterol derivative) constitutes about 32 mol% to about 37 mol% (e.g., about 34.3 mol%) of the total lipid present in the particle), and the PEG-lipid is a 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 phospholipids (such as DPPC) and cholesterol (wherein the phospholipid comprises about 15 mol% to about 25 mol% (e.g., about 20 mol%) of the total lipid present in the particle, and the cholesterol (or cholesterol derivative) comprises about 15 mol% to about 25 mol% (e.g., about 20 mol%) of the total lipid present in the particle), and the PEG-lipid is a PEG-DAA (e.g., PEG-cDMA). While these are preferred embodiments of the 1:57 formulation, one of skill in the art will understand that other cationic lipids, non-cationic lipids (including other phospholipids and other cholesterol derivatives), and conjugated lipids can be used in the 1:57 formulations described herein.

[0111] In a preferred embodiment, the 1:62 LNP formulation is a three-component system that does not contain phospholipids and is composed of about 1.5 mol% PEG-cDMA (or PEG-IDSA), about 61.5 mol% DLinDMA (or XTC2), and about 36.9 mol% cholesterol (or a derivative thereof). In another preferred embodiment, the 1:57 LNP formulation is a four-component system that is composed of about 1.4 mol% PEG-cDMA (or PEG-cDSA), about 57.1 mol% DLinDMA (or XTC2), about 7.1 mol% DPPC, and about 34.3 mol% cholesterol (or a derivative thereof). In yet another preferred embodiment, the 1:57 LNP formulation is a four-component system that is composed of about 1.4 mol% PEG-cDMA (or PEG-cDSA), about 57.1 mol% DLinDMA (or XTC2), about 20 mol% DPPC, and about 20 mol% cholesterol (or a derivative thereof). It is understood that these LNP formulations are targeted formulations and that the amount of lipids (both cationic and non-cationic) present in the LNP formulations and the amount of lipid conjugates present can vary.

[0112] The present invention also provides pharmaceutical compositions comprising a lipid particle (eg, LNP) described herein and a pharma- ceutically acceptable carrier.

[0113] In a further aspect, the present invention provides a method for introducing one or more active or therapeutic agents (e.g., nucleic acids) into a cell, comprising contacting the cell with a lipid particle (e.g., LNP) as described herein. In one embodiment, the cell is 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 or therapeutic agents (e.g., nucleic acids), comprising administering a lipid particle (e.g., LNP) as described herein to a mammalian subject. In a preferred embodiment, the mode of administration includes, but is not limited to, oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, and intradermal. Preferably, the mammalian subject is a human.

[0114] In one embodiment, at least about 5%, 10%, 15%, 20%, or 25% of the total injected dose of lipid particles (e.g., LNPs) is present in plasma at about 8, 12, 24, 36, or 48 hours after injection. In other embodiments, more than about 20%, more than 30%, more than 40%, and even about 60%, 70%, or 80% of the total injected dose of lipid particles (e.g., LNPs) is present in plasma at about 8, 12, 24, 36, or 48 hours after injection. In certain cases, more than about 10% of the particles are present in the plasma of the mammal at about 1 hour after administration. In certain other cases, the presence of lipid particles (e.g., LNPs) is detectable at least about 1 hour after administration of the particles. In certain embodiments, the presence of an active agent or therapeutic agent such as an interfering RNA (e.g., siRNA) or mRNA is detectable in cells (e.g., lung, liver, tumor, or inflammation site) about 8, 12, 24, 36, 48, 60, 72, or 96 hours after administration. In other embodiments, the downregulation of the expression of a target sequence by an active agent or therapeutic agent such as an interfering RNA (e.g., siRNA) is detectable about 8, 12, 24, 36, 48, 60, 72, or 96 hours after administration. In yet other embodiments, the downregulation of the expression of a target sequence by an active agent or therapeutic agent such as an interfering RNA (e.g., siRNA) occurs preferentially in tumor cells or in cells at inflammation sites. In further embodiments, the presence or action of an active agent or therapeutic agent such as an interfering RNA (e.g., siRNA) in cells at a site proximal or distal to the administration site or in 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, the upregulation of expression of a target sequence by an active agent or therapeutic agent such as an mRNA or self-amplifying RNA is detectable about 8, 12, 24, 36, 48, 60, 72, or 96 hours after administration. In yet other embodiments, the upregulation of expression of a target sequence by an active agent or therapeutic agent such as an mRNA or self-amplifying RNA occurs preferentially in tumor cells or in cells at sites of inflammation.In further embodiments, the presence or effect of an active or therapeutic agent, such as mRNA or self-replicating RNA, in cells at sites proximal or distal to the site of administration or in cells of the lung, liver, or tumor is detectable about 12, 24, 48, 72, or 96 hours, 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 present invention are administered parenterally or intraperitoneally.

[0115] In some embodiments, the lipid particles (e.g., LNPs) of the present invention are particularly useful in methods for therapeutic delivery of one or more nucleic acids, including interfering RNA sequences (e.g., siRNAs). In particular, it is an object of the present invention to provide in vitro and in vivo methods for treating diseases or disorders in mammals (e.g., rodents, such as mice, or primates, such as humans, chimpanzees, or monkeys) by downregulating or silencing the transcription and / or translation of one or more target nucleic acid sequences or genes of interest. As a non-limiting example, the methods of the present invention are useful for in vivo delivery of interfering RNA (e.g., siRNAs) to the liver and / or tumors of mammalian subjects. In certain embodiments, the disease or disorder is associated with expression and / or overexpression of a gene, and the expression or overexpression of the gene is reduced by the interfering RNA (e.g., siRNAs). In certain other embodiments, a therapeutically effective amount of lipid particles (e.g., LNPs) can be administered to a mammal. In some cases, the interfering RNA (e.g., siRNAs) are formulated into LNPs, and the particles are administered to a patient in need of such treatment. In other cases, cells are removed from the patient, interfering RNA (eg, siRNA) is delivered in vitro (eg, using LNPs described herein), and the cells are re-infused into the patient.

[0116] In an additional aspect, the present invention provides lipid particles (e.g., LNPs) that contain asymmetric interfering RNA (aiRNA) molecules that silence expression of a target gene, and methods of using such particles to silence expression of a target gene.

[0117] In one embodiment, the aiRNA molecule comprises a double-stranded (duplex) region of about 10 to about 25 (base-paired) nucleotides in length, the aiRNA molecule comprises an antisense strand comprising 5' and 3' overhangs, and the aiRNA molecule is capable of silencing target gene expression.

[0118] In one embodiment, the aiRNA molecule comprises a double-stranded (duplex) region of about 12-20, 12-19, 12-18, 13-17, or 14-17 (base-paired) nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 (base-paired) nucleotides in length. In certain other cases, the 5' and 3' overhangs on the antisense strand comprise sequences that are complementary to the target RNA sequence, and may optionally further comprise 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.

[0119] In other embodiments, the aiRNA molecule comprises modified nucleotides selected from the group consisting of 2'OMe nucleotides, 2'F nucleotides, 2'-deoxy nucleotides, 2'-O-MOE nucleotides, LNA nucleotides, and mixtures thereof. In preferred embodiments, the aiRNA molecule comprises 2'OMe nucleotides. As non-limiting examples, the 2'OMe nucleotides may be selected from the group consisting of 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, and mixtures thereof.

[0120] In a related aspect, the invention provides lipid particles (e.g., LNPs) that contain microRNA (miRNA) molecules that silence expression of a target gene, and methods of using such compositions to silence target gene expression.

[0121] In one embodiment, the miRNA molecule is comprised of a length of about 15 to about 60 nucleotides, and the miRNA molecule is capable of silencing the expression of a target gene.

[0122] In certain cases, the miRNA molecule is comprised of about 15-50, 15-40, or 15-30 nucleotides in length, more typically about 15-25 or 19-25 nucleotides in length, and preferably about 20-24, 21-22, or 21-23 nucleotides in length. In preferred embodiments, the miRNA molecule is a mature miRNA molecule that targets an RNA sequence of interest.

[0123] In some embodiments, the miRNA molecule comprises modified nucleotides selected from the group consisting of 2'OMe nucleotides, 2'F nucleotides, 2'-deoxy nucleotides, 2'-O-MOE nucleotides, LNA nucleotides, and mixtures thereof. In preferred embodiments, the miRNA molecule comprises 2'OMe nucleotides. As a non-limiting example, the 2'OMe nucleotides may be selected from the group consisting of 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, and mixtures thereof.

[0124] In some embodiments, the lipid particles (e.g., LNPs) of the present invention are useful in methods for 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 diseases or disorders in mammals (e.g., rodents such as mice or primates such as humans, chimpanzees, or monkeys) via expression of one or more target proteins. As a non-limiting example, the methods of the present invention are useful for 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 as described herein), and the cells are reinfused into the patient.

[0125] In other embodiments, the mRNA molecule comprises modified nucleotides selected from the group consisting of 2'OMe nucleotides, 2'F nucleotides, 2'-deoxy nucleotides, 2'-O-MOE nucleotides, LNA nucleotides, and mixtures thereof. In a related aspect, the invention provides lipid particles (e.g., LNPs) that contain microRNA (miRNA) molecules that silence expression of a target gene, and methods of silencing target gene expression using such compositions.

[0126] Thus, the lipid particles (e.g., LNPs) of the present invention are advantageous and suitable for use in administering active or therapeutic agents, such as nucleic acids (e.g., interfering RNA such as siRNA, aiRNA, and / or miRNA, or mRNA) to a subject (e.g., a mammal such as a human) because they are stable in the circulation, are of the size required for pharmacodynamic behavior that provides access to extravascular sites, and are capable of reaching target cell populations.

[0127] Activator Active agents (e.g., therapeutic agents) include any molecule or compound capable of exerting a desired effect on a cell, tissue, organ, or subject. Such effects can be, for example, biological, physiological, and / or cosmetic effects. Active agents 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, but are not limited to, antibodies (e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments; humanized antibodies, recombinant antibodies, recombinant human antibodies, Primatized™ antibodies), cytokines, growth factors, apoptotic factors, differentiation inducers, cell surface receptors and their ligands, hormones, and mixtures thereof. Examples of small molecules include, but are not limited to, small organic molecules or compounds, such as any conventional drug or drug known to those of skill in the art.

[0128] In some embodiments, the active agent is a therapeutic agent, or a salt or derivative thereof. The therapeutic agent derivative may be therapeutically active itself 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.

[0129] nucleic acid In certain embodiments, the lipid particles of the present invention are associated with nucleic acids to form nucleic acid-lipid particles (e.g., LNPs). In some embodiments, the nucleic acid is fully encapsulated in the lipid particle. As used herein, the term "nucleic acid" includes any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides generally referred to as oligonucleotides, and longer fragments referred to as polynucleotides. In certain embodiments, the oligonucleotides of the present invention are about 15 to about 60 nucleotides in length. Nucleic acids can be administered alone in lipid particles of the present invention or in combination (e.g., co-administration) with lipid particles of the present invention that include small molecules such as peptides, polypeptides, or conventional drugs.

[0130] In the context of the present invention, the terms "polynucleotide" and "oligonucleotide" refer to a polymer or oligomer of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars and intersugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers containing non-naturally occurring monomers or portions thereof which function similarly. Such modified or substituted oligonucleotides are often preferred over natural forms due to properties such as, for example, improved cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0131] Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleotides. Deoxyribooligonucleotides consist of a five-carbon sugar called deoxyribose, covalently linked to phosphates at the 5' and 3' carbons of this sugar to form an alternating, unbranched polymer. Ribooligonucleotides consist of a similar repeating structure in which the five-carbon sugar is ribose.

[0132] The nucleic acid present in the lipid-nucleic acid particle according to the present invention includes any form of nucleic acid known in the art. The nucleic acid used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrid. Examples of double-stranded DNA are described herein, and include, for example, structural genes, genes including control 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.

[0133] The nucleic acids of the present invention may be of various lengths, generally depending on the particular form of the nucleic acid. For example, in certain embodiments, the plasmid or gene may be about 1,000 to about 100,000 nucleotide residues in length. In certain embodiments, the oligonucleotides may range from about 10 to about 100 nucleotides in length. In various related embodiments, the single-stranded, double-stranded, and triple-stranded oligonucleotides may range in length from 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.

[0134] In certain embodiments, the oligonucleotide (or a strand thereof) of the present invention specifically hybridizes to or is complementary to a target polynucleotide sequence. The terms "specifically hybridizable" and "complementary" as used herein refer to 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 does not need to be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. In a preferred embodiment, an oligonucleotide is specifically hybridizable and has a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, or under conditions under which the assay is performed in the case of in vitro assays, if the binding of the oligonucleotide to the target sequence would disrupt the normal function of the target sequence, causing loss of utility or expression from the target sequence. Thus, an oligonucleotide may contain one, two, three, or more base substitutions compared to the region of the gene or mRNA sequence to which it targets or to which it specifically hybridizes.

[0135] siRNA The siRNA component of the nucleic acid-lipid particles of the present invention is capable of silencing the expression of a target gene of interest. Each strand of the siRNA duplex is typically about 15 to about 60 nucleotides long, preferably about 15 to about 30 nucleotides long. In certain embodiments, the siRNA comprises at least one modified nucleotide. Modified siRNAs are generally less immunostimulatory than the corresponding unmodified siRNA sequences and retain RNAi activity against a target gene of interest. In some embodiments, the modified siRNA contains at least one 2'OMe purine or pyrimidine nucleotide, such as a 2'OMe-guanosine nucleotide, a 2'OMe-uridine nucleotide, a 2'OMe-adenosine nucleotide, and / or a 2'OMe-cytosine nucleotide. In preferred embodiments, one or more of the uridine and / or guanosine nucleotides are modified. The modified nucleotide may be present 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).

[0136] Modified siRNAs generally contain from about 1% to about 100% (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in the double-stranded region of the siRNA duplex. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the nucleotides in the double-stranded region of the siRNA comprise modified nucleotides.

[0137] 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 in the double-stranded region of the siRNA comprise modified nucleotides.

[0138] In other embodiments, about 1% to about 25% (e.g., about 1% to 25%, 2% to 25%, 3% to 25%, 4% to 25%, 5% to 25%, 6% to 25%, 7% to 25%, 8% to 25%, 9% to 25%, 10% to 25%, 11% to 25%, 12% to 25%, 13% to 25%, 14% to 25%, 15% to 25%, 16% to 25%, 17% to 25%, 18% to 25%, 19% to 25%, 20% to 25%, 21% to 25%, 22% to 25%, 23% to 25%, 24% to 25%, etc.) or about 1% to about 20% (e.g., about 1% to 2 0%, 2%~20%, 3%~20%, 4%~20%, 5%~20%, 6%~20%, 7%~20%, 8%~20%, 9%~20%, 10%~20%, 11%~20%, 12%~20%, 13%~20%, 14%~20%, 15%~20%, 16%~20%, 17%~20%, 18%~20%, 19%~20%, 20%~20%, 21%~20%, 22%~20%, 23%~20%, 24%~20%, 25%~20%, 26%~20%, 27%~20%, 28%~20%, 29%~20%, 30%~20%, 31%~20%, 32%~20%, 33%~20%, 34% %~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.) contain modified nucleotides.

[0139] In further embodiments, for example, when one or both strands of the siRNA are selectively modified at uridine and / or guanosine nucleotides, the resulting modified siRNA contains 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 3%. % or less than about 1% modified nucleotides), or about 1% to about 30% modified nucleotides (e.g., 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%, 29% to 30%, 30% to 30%, 31% to 30%, 32% to 30%, 33% to 30%, 34% to 30%, 35% to 30%, 36% to 30%, 37% to 30%, 38% to 30%, 39% to 40%, 40% to 40%, 41% to 40%, 42% to 40%, 43% to 40%, 44% to 40%, 45% to 40%, 46% to 40%, 47% to 40%, 48% to 40%, 49% to 50%, 50% to 50%, 51% to 50%, 52% to 50%, 53% to 50%, 54% to 5 4%-30%, 15%-30%, 16%-30%, 17%-30%, 18%-30%, 19%-30%, 20%-30%, 21%-30%, 22%-30%, 23%-30%, 24%-30%, 25%-30%, 26%-30%, 27%-30%, 28%-30%, or 29%-30% modified nucleotides).

[0140] Selection of siRNA sequences Suitable siRNA sequences can be identified using any means known in the art, typically by combining the methods described in Elbashir et al., Nature, 411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001) with the rational design rules described in Reynolds et al., Nature Biotech., 22(3):326-330 (2004).

[0141] Generally, the nucleotide sequence 3' of the AUG start codon of the transcript from the target gene of interest is scanned for dinucleotide sequences (e.g., AA, NA, CC, GG, or UU (N=C, G, or U)) (see, e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). The nucleotide immediately 3' of the dinucleotide sequence is identified as a potential siRNA sequence (i.e., target sequence or sense strand sequence). Usually, 19, 21, 23, 25, 27, 29, 31, 33, 35, or more nucleotides immediately 3' of the dinucleotide sequence are identified as a potential siRNA sequence. In some embodiments, the dinucleotide sequence is an AA or NA sequence, and 19 nucleotides immediately 3' of 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 increase the silencing efficiency of the siRNA sequence, potential siRNA sequences can be analyzed to identify sites that do not contain regions of homology with other coding sequences, for example in the target cell or organism. For example, a suitable siRNA sequence of about 21 base pairs will usually not have more than 16-17 consecutive base pairs that are homologous to coding sequences in the target cell or organism. If the siRNA sequence is to be expressed from an RNA PolIII promoter, select an siRNA sequence that lacks more than four consecutive A's or T's.

[0142] After identifying potential siRNA sequences, complementary sequences (i.e., antisense strand sequences) can be designed. Potential siRNA sequences can also be analyzed using various criteria known in the art. For example, to enhance their silencing efficiency, siRNA sequences can be analyzed by rational design algorithms to identify sequences that have one or more of the following characteristics: (1) a G / C content that is about 25% to about 60% G / C, (2) at least three A / Us at positions 15-19 of the sense strand, (3) no internal repeats, (4) an A at position 19 of the sense strand, (5) an A at position 3 of the sense strand, (6) a U at position 10 of the sense strand, (7) position 19 of the sense strand is not G / C, and (8) position 13 of the sense strand is not G. siRNA design tools that assign appropriate values ​​for each of these characteristics and incorporate algorithms useful for selecting siRNAs can be found, for example, at http: / / boz094.ust.hk / RNAi / siRNA. One of skill in the art will appreciate that sequences having one or more of the aforementioned characteristics can be selected for further analysis and testing as potential siRNA sequences.

[0143] In addition, potential siRNA sequences that have one or more of the following criteria are often excluded as siRNA: (1) sequences that contain 4 or more consecutive identical bases, (2) sequences that contain homopolymers of G (i.e., to reduce possible non-specific effects due to the structural properties of these polymers), (3) sequences that contain triple base motifs (e.g., GGG, CCC, AAA, or TTT), (4) sequences that contain 7 or more consecutive G / Cs, and (5) sequences that contain 4 or more direct repeats of bases within the candidate, resulting in internal foldback structure.However, those skilled in the art will understand that sequences that have one or more of the above characteristics can still be selected as potential siRNA sequences for further analysis and testing.

[0144] In some embodiments, potential siRNA sequences can be further analyzed based on siRNA duplex asymmetry, for example, 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 secondary structure at the target site, for example, as described in Luo et al., Biophys.Res.Commun., 318:303-310 (2004). For example, secondary structure at the target site can be modeled using the Mfold algorithm (available at http: / / www.bioinfo.rpi.edu / applications / mfold / rna / form1.cgi), and siRNA sequences with low secondary structure and stem loop in base pairing form that favor accessibility at the target site can be selected.

[0145] After identifying potential siRNA sequences, the sequences can be analyzed for the presence of any immunostimulatory properties, for example, using in vitro cytokine assays or in vivo animal models. Motifs within the sense and / or antisense strands of the siRNA sequence, such as GU-rich motifs (e.g., 5'-GU-3', 5'-UGU-3', 5'-GUGU-3', 5'-UGUGU-3', etc.), can also provide an indication of whether the sequence may be immunostimulatory. If an siRNA molecule is found to be immunostimulatory, it can then be modified to reduce immunostimulatory properties, as described herein. As a non-limiting example, to determine whether an siRNA is an immunostimulatory siRNA or a non-immunostimulatory siRNA, the siRNA sequence can be contacted with mammalian responder cells under conditions that cause the cells to produce a detectable immune response. The mammalian responder cells can be derived from a naive mammal (i.e., a mammal that has not previously been contacted with 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 may 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 of the sense strand and / or antisense strand with modified nucleotides 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 in the double-stranded region of the siRNA duplex can be replaced with modified nucleotides, such as 2'OMe nucleotides. The modified siRNA can then be contacted with mammalian responder cells as described above to confirm that its immunostimulatory properties are reduced or suppressed.

[0146] Suitable in vitro assays for detecting immune responses include the double monoclonal antibody sandwich immunoassay method of David et al. (U.S. Pat. No. 4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970)); the "Western blot" method of Gordon et al. (U.S. Pat. No. 4,452,901); immunoprecipitation of labeled ligands (Brown et al., J. Biol. Chem., 255:4980-4983 (1980)); enzyme-linked immunosorbent assays (ELISAs), e.g., as described by Raines et al., J. Biol. Chem., 257:5154-5160 (1982); immunocytochemistry methods involving the use of fluorescent dyes (Brooks et al., J. Biol. Chem., 257:5154-5160 (1982)); 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)). In addition to the foregoing immunoassays, numerous 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 incorporated herein by reference in their entireties for all purposes.

[0147] Non-limiting examples of in vivo models for detecting immune responses include the in vivo mouse cytokine induction assay described, for example, in Judge et al., Mol. Ther., 13:494-505 (2006). In certain embodiments, the assay can be performed as follows: (1) siRNA can be administered into the lateral tail vein by standard intravenous injection, (2) blood can be collected by cardiac puncture about 6 hours after administration and processed as plasma for cytokine analysis, and (3) cytokines can be quantified using sandwich ELISA kits according to the manufacturer's instructions (e.g., mouse and human IFN-α (PBL Biomedical; Piscataway, NJ), human IL-6 and TNF-α (eBioscience; San Diego, Calif.), and mouse IL-6, TNF-α, and IFN-γ (BD Biosciences; San Diego, Calif.)).

[0148] Monoclonal antibodies that specifically bind to cytokines and growth factors are commercially available from several sources and can be produced 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)). 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, the monoclonal antibody is labeled to facilitate detection (e.g., with any composition detectable by spectroscopic, photochemical, biochemical, electrical, optical, or chemical means).

[0149] Creation of siRNA molecules siRNA can be provided in several forms, including, 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. siRNA sequence can have overhang (e.g., 3' or 5' overhang as described in Elbashir et al., Genes Dev., 15:188 (2001) or Nykanen et al., Cell, 107:309 (2001)), or can lack overhang (i.e., have blunt ends).

[0150] A population of RNA can be used to obtain long precursor RNA, or long precursor RNA with substantial or complete identity to a selected target sequence can be used to generate siRNA. RNA can be isolated from cells or tissues, synthesized, and / or cloned according to methods well known to those skilled in the art. RNA can be a mixed population (obtained from cells or tissues, transcribed from cDNA, subtracted, selected, etc.), or can represent a single target sequence. RNA can be natural (e.g., isolated from tissue or cell samples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCR products or cloned cDNA), or chemically synthesized.

[0151] In order to form long dsRNA, in the case of synthetic RNA, the complement is also transcribed in vitro and hybridized to form dsRNA.When using 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 (for example, to form dsRNA for digestion by E.coli RNAseIII or Dicer).The precursor RNA is then hybridized to form double-stranded RNA for digestion.The dsRNA can be directly administered to the subject or digested in vitro before administration.

[0152] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, creating and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra), as is the PCR method (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)). Expression libraries are also well known to those of skill in the art. Further fundamental 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 which are incorporated herein by reference in their entireties for all purposes.

[0153] Preferably, siRNA is chemically synthesized. Oligonucleotides, including 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). Synthesis of oligonucleotides uses common nucleic acid protecting 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 performed on a 96-well plate synthesizer from Protogene (Palo Alto, Calif.). However, larger or smaller scale syntheses are also within the scope of the invention. Reagents suitable for oligonucleotide synthesis, methods of RNA deprotection, and methods of RNA purification are known to those of skill in the art.

[0154] 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 provide separate fragments or strands that hybridize to form an siRNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. Tandem synthesis of siRNA can be easily adapted to both multi-well / multi-plate synthesis platforms and large-scale synthesis platforms using batch reactors, synthesis columns, etc. Alternatively, siRNA molecules can be assembled from two separate oligonucleotides, one oligonucleotide containing the sense strand of siRNA and the other containing the antisense strand. For example, each strand can be synthesized separately and linked to each other 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 the self-complementary sense and antisense regions hybridize to form an siRNA duplex with a hairpin secondary structure.

[0155] Modification of siRNA sequence In certain aspects, the siRNA molecule comprises a duplex having two strands and at least one modified nucleotide in the double-stranded region, where each strand is 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 expression of the target sequence. In preferred embodiments, the degree of chemical modification introduced into the siRNA molecule strikes a balance between reducing or suppressing the immunostimulatory properties of the siRNA and retaining RNAi activity. As a non-limiting example, an siRNA molecule targeting a gene of interest may be minimally modified (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) at selective uridine and / or guanosine nucleotides within the siRNA duplex to eliminate immune responses generated by the siRNA while retaining its ability to silence target gene expression.

[0156] The modified nucleotide examples suitable for use in the present invention include, but are not limited to, ribonucleotides with 2'-O-methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), 2'-deoxy, 5-C-methyl, 2'-O-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino or 2'-C-allyl group.Modified nucleotides with 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 include one or more G-clamp nucleotides. G-clamp nucleotides refer to modified cytosine analogs whose modification confers the ability to hydrogen bond to both the Watson-Crick and Hoogsteen faces of complementary guanine nucleotides in a duplex (see, e.g., Lin et al., J. Am. Chem. Soc., 120:8531-8532 (1998)). Additionally, nucleotides having nucleotide base analogs, such as C-phenyl, C-naphthyl, and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives, such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucl. Acids Res., 29:2437-2447 (2001)), can be incorporated into the siRNA molecules.

[0157] 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'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3',4'-seconucleotides, 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, These include, but are not limited to, 3'-5'-inverted deoxy abasic moieties, 5'-amino-alkyl phosphates, 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 1,4-butanediol phosphate, 3'-phosphoramidates, 5'-phosphoramidates, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 5'-amino, 3'-phosphorothioates, 5'-phosphorothioates, phosphorodithioates, and bridged or unbridged methylphosphonate or 5'-mercapto moieties (see, e.g., U.S. Pat. No. 5,998,203; Beaucage et al., Tetrahedron 49:1925 (1993)).Non-limiting examples of phosphate backbone modifications (i.e., resulting in modified internucleotide linkages) include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitutions (see, for example, Hunziker et al., Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417 (1995); Mesmaeker et al., Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39 (1994)). Such chemical modifications can occur at the 5'-end and / or 3'-end of the sense strand, antisense strand, or both strands of the siRNA. The disclosures of these references are incorporated by reference herein in their entireties for all purposes.

[0158] In some embodiments, the sense strand and / or antisense strand of the siRNA molecule may further comprise a 3'-end overhang having about 1 to about 4 (e.g., 1, 2, 3, or 4) 2'-deoxyribonucleotides and / or any combination of modified and unmodified nucleotides. Further examples of the types of modified nucleotides and chemical modifications that can be introduced into the siRNA molecule are described, for example, in British Patent No. GB2,397,818B and US Patent Publication Nos. 20040192626, 20050282188, and 20070135372, the disclosures of which are incorporated by reference herein in their entireties for all purposes.

[0159] 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 that contain sugar and / or phosphate substitutions, allowing the remaining bases to exhibit their activity.This group or compound does not contain a commonly recognized nucleotide base such as adenosine, guanine, cytosine, uracil, or thymine, and is therefore abasic in that it lacks a base at the 1' position.

[0160] In other embodiments, chemical modification of siRNA comprises binding a conjugate to siRNA molecule.The conjugate can be bound to the 5'-end and / or 3'-end of the sense strand and / or antisense strand of siRNA via a covalent bond such as a biodegradable linker.The conjugate can also be bound to siRNA via a carbamate group or other linking group (see, for example, US Patent Application Publication Nos. 20050074771, 20050043219, and 20050158727).In certain cases, the conjugate is a molecule that facilitates the delivery of siRNA to cells. Examples of conjugate molecules suitable for 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), sugars (e.g., galactose, galactosamine, N-acetylgalactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands for cellular 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, cleavage agents, and crosslinker conjugate molecules described in US Patent Publication Nos. 20050119470 and 20050107325. Still other examples include 2'-O-alkylamines, 2'-β-alkoxyalkylamines, polyamines, C5-cationically modified pyrimidines, cationic peptides, guanidinium groups, amidininium groups, cationic amino acid conjugate molecules described in US Patent Publication No. 20050153337. Additional examples include hydrophobic group, membrane active compound, cell permeable compound, cell targeting signal, interaction modifier, and steric stabilizer conjugate molecules described in US Patent Publication No. 20040167090. Further examples include conjugate molecules described in US Patent Publication No. 20050239739. The type of conjugate used and the degree of conjugation with siRNA molecules can be evaluated for improved siRNA pharmacokinetic profile, bioavailability, and / or stability while retaining RNAi activity. Thus, one skilled 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 with various conjugates attached to identify those with improved properties and full RNAi activity. The disclosures of the aforementioned patent documents are incorporated herein by reference in their entirety for all purposes.

[0161] Target Gene In certain embodiments, the nucleic acid component (e.g., siRNA) of the nucleic acid-lipid particle described herein can be used to downregulate or silence the translation (i.e., expression) of a gene of interest. Genes of interest include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., liver diseases and liver damage), genes associated with tumorigenesis and cell transformation (e.g., cancer), angiogenesis genes, immune regulatory 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.

[0162] Genes associated with viral infection and survival include genes expressed by viruses to bind, enter, and replicate within a cell. Of particular interest are viral sequences associated with chronic viral diseases. Viral sequences of particular interest include filoviruses, such as Ebola virus and Marburg virus (see, e.g., Geisbert et al., J. Infect. Dis., 193:1650-1657 (2006)), arenaviruses, such as Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus (see, e.g., 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, e.g., Steinhauer et al., Annu Rev Genet., 36:305-332 (2002), and Neumann et al., J Gen Virol., 83:2635-2662 (2002)), hepatitis viruses (see, e.g., Hamasaki et al., FEBS Lett., 543:51 (2003); Yokota et al., EMBO Rep., 4:602 (2003); Schlomai et al., Hepatology, 37:764 (2003); Wilson et al., Proc. 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.,77:7174(2003); Stephenson,JAMA,289:1494(2003); Qin et al.,Proc.Natl.Acad.Sci.USA,100:183(2003)), herpesvirus (Jia et al.,J.Virol.,77:3301(2003)), and human papillomavirus (HPV) (Hall et al.,J.Virol.,77:6066(2003); Jiang et al.,Oncogene,21:6041(2002)).

[0163] Exemplary filovirus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences encoding structural proteins (e.g., VP30, VP35, nucleoprotein (NP), polymerase protein (L-pol)) and membrane-associated proteins (e.g., VP40, glycoprotein (GP), VP24). The complete genome sequence of Ebola virus is described, for example, in Genbank Accession Nos. 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 Nos. U77385 and AY058897. The sequence of Ebola virus L-pol is described, for example, in Genbank Accession No. X67110. The sequence of Ebola virus VP40 is described, for example, in Genbank Accession No. AY058896. The sequence of Ebola virus NP is described, for example, in Genbank Accession No. AY058895. The sequence of Ebola virus GP is described, for example, in Genbank Accession No. AY058898, Sanchez et al., Virus Res., 29:215-240 (1993), Will et al., J. Virol., 67:1203-1210 (1993), Volchkov et al., FEBS Lett., 305:181-184 (1992), and U.S. Patent No. 6,713,069. Further Ebola virus sequences are described, for example, in Genbank Accession Nos. L11365 and X61274. The complete genome sequence of Marburg virus is described, for example, in Genbank Accession Nos. NC_001608, AY430365, AY430366, and AY358025. The sequence of Marburg virus GP is described, for example, in Genbank Accession Nos. AF005734, AF005733, and AF005732.The sequence of Marburg virus VP35 is described, for example, in Genbank Accession Nos. AF005731 and AF005730. Additional Marburg virus sequences are described, for example, in Genbank Accession Nos. X64406, Z29337, AF005735, and Z12132. Non-limiting examples of siRNA molecules targeting Ebola and Marburg virus nucleic acid sequences include those described in U.S. Patent Application Publication No. 20070135370, the disclosure of which is incorporated by reference in its entirety for all purposes.

[0164] 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), nonstructural proteins (NS1 and NS2), RNA polymerase (PA, PB1, PB2), neuraminidase (NA), and hemagglutinin (HA). Influenza A NP sequences can be found, for example, in Genbank Accession Nos. 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. Influenza A PA sequences are available, for example, from Genbank accession numbers AY818132, AY790280, AY646171, AY818132, AY818133, AY646179, AY818134, AY551934, AY651613, AY651610, AY651620, AY651617, AY651600, A 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 incorporated by reference in its entirety for all purposes.

[0165] Exemplary hepatitis virus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences involved in transcription and translation (e.g., En1, En2, X, P) and nucleic acid sequences encoding structural proteins (e.g., core protein, including C protein and C-related protein, capsid and envelope proteins, including S, M, and / or L proteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY, supra). Exemplary hepatitis C virus (HCV) nucleic acid sequences that can be silenced include, but are not limited to, 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. 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 Hepatitis A virus nucleic acid sequence is described, for example, in Genbank Accession No. NC_001489, the Hepatitis B virus nucleic acid sequence is described, for example, in Genbank Accession No. NC_003977, the Hepatitis D virus nucleic acid sequence is described, for example, in Genbank Accession No. NC_001653, the Hepatitis E virus nucleic acid sequence is described, for example, in Genbank Accession No. NC_001434, and the Hepatitis G virus nucleic acid sequence is described, for example, in Genbank Accession No. NC_001710.Silencing the sequence that codes for the gene related to viral infection and survival can be advantageously used in combination with the administration of conventional drugs used to treat viral conditions.Non-limiting examples of siRNA molecules that target 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 on March 20, 2009, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0166] Genes associated with metabolic diseases and disorders (e.g., disorders in which the liver is a target, and liver diseases and disorders) include, for example, genes expressed in dyslipidemia (e.g., liver X receptors such as LXRα and LXRβ (Genbank Accession No. NM_007121), farnesoid X receptor (FXR) (Genbank Accession No. 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 No. NM_000384), apolipoprotein CIII (ApoC3) (Genbank Accession Nos. NM_000040 and NG_008949) REGION:5001.8164), and apolipoprotein E (ApoE) (Genbank accession numbers NM_000041 and NG_007084 REGION:5001.8612), and diabetes (e.g., glucose 6-phosphatase) (e.g., Forman et al., Cell, 81:687 (1995); Seol et al., Mol. Endocrinol., 9:72 (1995); Zavacki et al., Proc. Natl. Acad. Sci. USA, 94:7909 (1997); Sakai et al., Cell, 85:1037-1046 (1996); Duncan et al., J. Biol. Chem., 272:12778-12785 (1997); Willy et al., Genes 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 appreciate that genes associated with metabolic diseases and disorders (e.g., disorders in which the liver is a target, as well as liver diseases and disorders) include genes expressed in the liver itself as well as genes expressed in other organs and tissues.Silencing of sequences that code for genes related to metabolic diseases and disorders can be advantageously used in combination with the administration of conventional drugs used to treat the disease or disorder.Non-limiting examples of siRNA molecules that target ApoB genes include those described in US Patent Publication No. 20060134189, the disclosure of which is incorporated herein by reference in its entirety for all purposes.Non-limiting examples of siRNA molecules that target ApoC3 genes include those described in US Provisional Patent Application No. 61 / 147,235, filed on January 26, 2009, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0167] Examples of gene sequences associated with tumorigenesis and cell transformation (e.g., cancer or other neoplasia) include mitotic kinesins such as Eg5 (KSP, KIF11; Genbank Accession No. NM_004523); serine / threonine kinases such as polo-like kinase 1 (PLK-1) (Genbank Accession No. NM_005030, Barr et al., Nat. Rev. Mol. Cell. Biol., 5:429-440 (2004)); tyrosine kinases such as WEE1 (Genbank Accession Nos. NM_003390 and NM_001143976); apoptosis inhibitors such as XIAP (Genbank Accession No. NM_001167); CSN1, CSN2, CSN3, CSN4, CSN5 (JAB1; Genbank Accession No. NM_00683 7), COP9 signalosome subunits such as CSN6, CSN7A, CSN7B and CSN8; ubiquitin ligases such as COP1 (RFWD2; Genbank Accession Nos. NM_022457 and NM_001001740); and histone deacetylases such as HDAC1, HDAC2 (Genbank Accession Nos. NM_001527), HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, etc. 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 incorporated herein by reference in its entirety for all purposes. Non-limiting examples of siRNA molecules targeting the PLK-1 gene include those described in U.S. Patent Application Publication Nos. 20050107316 and 20070265438, and U.S. Patent Application No. 12 / 343,342, filed December 23, 2008, the disclosures of which are 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 disclosures of which are incorporated by reference in their entirety for all purposes.

[0168] Further examples of gene sequences associated with tumorigenesis and cell transformation include translocation sequences, e.g., MLL fusion genes, BCR-ABL (Wilda et al., Oncogene, 21:5716 (2002); Scherr et al., Blood, 101:1566 (2003)), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO, and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003)); overexpressed sequences, e.g., multidrug resistance genes (Nieth et al., FEBS Lett., 545:144 (2003); Wu et al., Cancer Res. 63:1515 (2003)), cyclins (Li et al., Cancer Res., 63:3593 (2003); Zou et al., Cancer Res. 63:3593 (2003)), and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003)). al., Genes Dev., 16:2923(2002)), beta-catenin (Verma et al., Clin Cancer Res., 9:1291(2003)), telomerase gene (Kosciolek et al., Mol Cancer Ther., 2:209(2003)), c-MYC, N-MYC, BCL-2, growth factor receptors (e.g., EGFR / ErbB1 (Genbank accession numbers NM_005228, NM_201282, NM_201283, and NM_201284; see also Nagy et al. Exp. Cell 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2060, 2061, 2062, 2063, 2064, Res., 285:39-49 (2003)), ErbB2 / HER-2 (Genbank Accession Nos. NM_004448 and NM_001005862), ErbB3 (Genbank Accession Nos. NM_001982 and NM_001005915), and ErbB4 (Genbank Accession Nos. NM_005235 and NM_001042599)); and 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 Ser. No. 11 / 807,872, filed May 29, 2007, the disclosure of which is incorporated by reference in its entirety herein for all purposes.

[0169] 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 associated with tumor migration are also target sequences of interest, e.g., integrins, selectins, and metalloproteinases. The foregoing examples are not exclusive. One skilled in the art will understand that any whole or partial gene sequence that promotes or drives tumorigenesis or cell transformation, tumor growth, or tumor migration may be included as a template sequence.

[0170] Angiogenic genes can promote the formation of new blood vessels. Of particular interest is 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, US Patent Publication No. 20040142895, and CA2456444, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0171] Antiangiogenic genes can inhibit neovascularization. These genes are particularly useful for treating cancers in which angiogenesis plays a role in the pathological development of the disease. Examples of antiangiogenic genes include, but are not limited to, endostatin (see, e.g., U.S. Pat. No. 6,174,861), angiostatin (see, e.g., U.S. Pat. No. 5,639,725), and VEGFR2 (see, e.g., Decaussin et al., J. Pathol., 188:369-377 (1999)), the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0172] Immunomodulatory genes are genes that regulate one or more immune responses. Examples of immunomodulatory genes include, but are not limited to, 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. Fas and Fas ligand genes are also immunomodulatory target sequences of interest (Song et al., Nat.Med., 9:347 (2003)). Genes encoding second signaling molecules in hematopoietic and lymphoid cells, for example, 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.

[0173] Cell receptor ligands include ligands that can bind to cell surface receptors (e.g., insulin receptors, EPO receptors, G protein-coupled receptors, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.) and regulate (e.g., inhibit, activate, etc.) physiological pathways in which the receptor is involved (e.g., regulation of glucose levels, blood cell development, mitogenesis, etc.). Examples of cell receptor ligands include, but are not limited to, cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G protein-coupled receptor ligands, etc. Templates encoding expansions of trinucleotide repeats (e.g., CAG repeats) are used to silence pathogenic sequences in neurodegenerative disorders caused by expansions of trinucleotide repeats, such as spinal and bulbar muscular atrophy and Huntington's disease (Caplen et al., Hum. Mol. Genet., 11:175 (2002)).

[0174] Other specific target genes that can be targeted by nucleic acids (e.g., by siRNA) to downregulate or silence expression of the genes include aortic smooth muscle alpha 2 actin (ACTA2), alcohol dehydrogenase 1A (ADH1A), alcohol dehydrogenase 4 (ADH4), alcohol dehydrogenase 6 (ADH6), afamin (AFM), angiotensinogen (AGT), serine-pyruvate aminotransferase (AGXT), 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 C3 (APOC3), apolipoprotein C-IV (APOC4), apolipoprotein A-II (APOA2), apolipoprotein B-II (APOC5), apolipoprotein B-IV (APOC6), apolipoprotein B-II (APOC7), apolipoprotein B-II (APOC8), apolipoprotein B-II (APOC9), apolipoprotein B-II (APOC10), apolipoprotein B-II (APOC11), apolipoprotein B-II (APOC12), apolipoprotein B-II (APOC13), apolipoprotein B-II (APOC14), apolipoprotein B-II (APOC15), apolipoprotein B-II (APOC16), apolipoprotein B-II (APOC17), apolipoprotein B-II (APOC18), apolipoprotein B-II (APOC19), apolipoprotein B-II (APOC19), apolipoprotein B-II (APOC19), apolipoprotein B-II (APOC19), apolipoprotein B-II (APOC10), apolipoprotein B-II (APOC11), apolipoprotein B-II (APOC12), apolipoprotein B-II (APOC13), apolipoprotein B-II (APOC14), apolipoprotein B-II (APOC15), apolipoprotein B-II (APOC16), apolipoprotein B-II (APOC17), apolipoprotein B-II (APOC18), apolipoprotein B-II (APOC19), apolipoprotein B-II (APOC19), a Protein F (APOF), beta-2-glycoprotein 1 (APOH), aquaporin-9 (AQP9), bile acid-CoA:amino acid N-acyltransferase (BAAT), C4b-binding protein beta chain (C4BPB), 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 C8 alpha chain (C8G), complement component C8 beta chain (C8G), complement component C8 gamma chain (C8H), complement component C8 alpha chain (C8H), complement component C8 beta chain (C8H ... alpha chain (C8H), complement component C8 beta chain (C8H), complement component C8 gamma chain (C8H), complement component C8 alpha chain (C8H), complement component C8 beta chain (C8H), complement component C8 alpha chain (C8H), complement component C8 beta chain (C8H), complement component C8 alpha chain (C8H), complement component C8 beta chain (C8H), complement component C8 alpha chain (C8H), G), complement component C9 (C9), calmodulin-binding transcription activator 1 (CAMTA1), CD38 (CD38), complement factor B (CFB), complement factor H-related protein 1 (CFHR1), complement factor H-related protein 2 (CFHR2), complement factor H-related protein 3 (CFHR3), cannabinoid receptor 1 (CNR1), ceruloplasmin (CP), carboxypeptidase B2 (CPB2), connective tissue growth factor (CTGF), CXC motif chemokine 2 (CXCL2), cytochrome P450 1A2 (CYP1A2), cytochrome P450 2A6 (CYP2A6), cytochrome P450 2C8 (CYP2C8), cytochrome P450 2C9 (CYP2C9), cytochrome P450 family 2 subfamily D member 6 (CYP2D6),Cytochrome P450 2E1 (CYP2E1), phylloquinone omega-hydroxylase CYP4F2 (CYP4F2), 7-alpha-hydroxycholest-4-en-3-one 12-alpha-hydroxylase (CYP8B1), dipeptidyl peptidase 4 (DPP4), coagulation factor 12 (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 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 (glycolate oxidase) 1 (HAO1), HGF activator (HGFAC), haptoglobin-related protein; haptoglobin (HPR), hemopexin (HPX), histidine lysate (HLA), and glycine N-methyltransferase (GNMT). Glucoprotein 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-derived chemotaxin 2 (LECT2), lipoprotein(a) (LPA), mannan-binding lectin serine peptidase 2 (MASP2), S-adenosylmethionine synthase isoform type 1 (MAT1A), NADPH oxidase 4 (NOX4), poly[ADP-ribose]polymerase 1 (PARP1), paraoxonase 1 (PON1), paraoxonase 3 (PON3), vitamin K-dependent protein C (PROC), retinol dehydrogenase 16 (RDH16),Constitutive serum amyloid A4 (SAA4), serine dehydratase (SDS), serpin family A member 1 (SERPINA1), serpin A11 (SERPINA11), kallistatin (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-facilitated glucose transporter These include, but are not limited to, sodium-coupled neutral amino acid 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 salt sulfotransferase (SULT2A1), tyrosine aminotransferase (TAT), tryptophan 2,3-dioxygenase (TDO2), UDP-glucuronosyltransferase 2 family polypeptide B10 (UGT2B10), UDP-glucuronosyltransferase 2 family polypeptide B15 (UGT2B15), UDP-glucuronosyltransferase 2 family polypeptide B4 (UGT2B4), and vitronectin (VTN).

[0175] In addition to their usefulness in silencing the expression of any of the aforementioned genes for therapeutic purposes, certain nucleic acids (e.g., siRNAs) described herein are also useful in research and development applications, as well as diagnostic, preventive, prognostic, clinical, and other health care applications. As a non-limiting example, certain nucleic acids (e.g., siRNAs) can be used in target validation studies aimed at testing whether a gene of interest is a potential therapeutic target. Certain nucleic acids (e.g., siRNAs) can also be used in target identification studies aimed at finding genes as potential therapeutic targets.

[0176] CRISPR Targeted genome editing has evolved from a niche technology to a method used by many biology researchers. This evolution has been greatly facilitated by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR) technology (see, for example, Sander et al., Nature Biotechnology, 32(4), 347-355, including Supplementary Information (2014); International Publication Nos. WO2016 / 197132 and 2016 / 197133). Thus, 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 regard to targets for using CRISPR, the guide RNA (gRNA) utilized in CRISPR technology can be designed to target a specifically identified sequence, for example, a target gene (e.g., a target gene in the HBV genome). Examples of such target sequences are shown in International Publication No. WO2016 / 197132. Furthermore, International Publication No. WO2013 / 151665 (see, e.g., Table 6, which is specifically incorporated by reference, particularly including Table 6 and the accompanying sequence listing) describes approximately 35,000 mRNA sequences claimed in association with mRNA expression constructs. Certain embodiments of the present invention utilize CRISPR technology to target the expression of any of these sequences. Certain embodiments of the present invention can also utilize CRISPR technology to target the expression of the target genes discussed herein.

[0177] aiRNA Similar to siRNA, asymmetric interfering RNA (aiRNA) can result in effective silencing of various genes in mammalian cells by recruiting the RNA-induced silencing complex (RISC) and 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 with a sense strand and an antisense strand, which contains overhangs at the 3' and 5' ends of the antisense strand. aiRNA is generally asymmetric because the sense strand is shorter at both ends compared to the complementary antisense strand. In some embodiments, aiRNA molecules can be designed, synthesized, and annealed under conditions similar to those used for siRNA molecules. As a non-limiting example, aiRNA sequences can be selected and generated using the methods described above for selecting siRNA sequences.

[0178] 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 with overhangs at the 3' and 5' ends of the antisense strand to target the mRNA of interest. 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, and preferably about 20-24, 21-22, or 21-23 nucleotides in length.

[0179] 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 contain one or more modified nucleotides, for example, in the double-stranded (duplex) region and / or in the antisense overhang. As non-limiting examples, the aiRNA sequence may contain one or more of the modified nucleotides described above for siRNA sequences. In preferred embodiments, the aiRNA molecule contains 2'OMe nucleotides, such as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, or mixtures thereof.

[0180] In certain embodiments, the aiRNA molecule may comprise an antisense strand that corresponds to the antisense strand of an siRNA molecule, e.g., one of the siRNA molecules described herein. In other embodiments, the aiRNA molecule may 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 tumorigenesis 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.

[0181] miRNA Generally, microRNAs (miRNAs) are single-stranded RNA molecules about 21-23 nucleotides long that control gene expression. Although miRNAs are encoded by genes from which the miRNAs are transcribed (non-coding RNA), miRNAs are not translated into proteins (non-coding RNAs); instead, each primary transcript (pri-miRNA) is processed into a short stem-loop structure called a pre-miRNA, which is finally processed into a functional mature miRNA. Mature miRNA molecules are either partially or fully complementary to one or more messenger RNA (mRNA) molecules, and their primary function is to downregulate gene expression. Identification of miRNA molecules has been 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.

[0182] Genes encoding miRNAs are much longer than the processed mature miRNA molecules. miRNAs are first transcribed as primary transcripts or pri-miRNAs with a cap and polyA tail, which are processed in the cell nucleus into short stem-loop structures of about 70 nucleotides known as pre-miRNAs. In animals, this processing is carried out by a protein complex known as the microprocessor complex, which consists of the nuclease Drosha and the double-stranded RNA-binding protein Pasha (Denli et al., Nature, 432:231-235 (2004)). These pre-miRNAs are then processed into mature miRNAs in the cytoplasm 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 DNA can serve as a template to generate miRNAs.

[0183] When Dicer cleaves the pre-miRNA stem loop, two complementary short RNA molecules are formed, only one of which 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 incorporation into the active RISC complex, miRNAs base pair with their complementary mRNA molecules and induce degradation and / or translational silencing of the target mRNA.

[0184] Mammalian miRNA molecules are usually complementary to a site within the 3'UTR of a target mRNA sequence. In certain cases, annealing of a miRNA to a target mRNA inhibits protein translation by blocking the protein translation machinery. In certain other cases, annealing of a miRNA to a target mRNA promotes cleavage and degradation of the target mRNA through a process similar to RNA interference (RNAi). miRNAs can also target methylation of genomic sites corresponding to the targeted mRNA. In general, miRNAs function in association with a complement of proteins collectively referred to as miRNPs.

[0185] In certain aspects, the miRNA molecules described herein are about 15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotides in length, more typically about 15-30, 15-25, or 19-25 nucleotides in length, and preferably about 20-24, 21-22, or 21-23 nucleotides in length. In certain other aspects, the miRNA molecules may include one or more modified nucleotides. As non-limiting examples, the miRNA sequence may include one or more of the modified nucleotides described above for siRNA sequences. In preferred embodiments, the miRNA molecules include 2'OMe nucleotides, such as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, or mixtures thereof.

[0186] In some embodiments, miRNA molecules 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 tumorigenesis and cell transformation, angiogenic genes, immune regulatory genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.

[0187] In other embodiments, one or more agents that block the activity of miRNA that targets mRNA of interest are administered using lipid particles (e.g., nucleic acid-lipid particles) of the present invention. Examples of blocking agents include, but are not limited to, steric blocking oligonucleotides, locked nucleic acid oligonucleotides, and morpholino oligonucleotides. Such blocking agents can directly bind to miRNA or miRNA binding sites on target mRNA.

[0188] Antisense oligonucleotides In one embodiment, the nucleic acid is an antisense oligonucleotide directed to a target gene or sequence of interest. The term "antisense oligonucleotide" or "antisense" includes oligonucleotides that are complementary to a targeted polynucleotide sequence. An antisense oligonucleotide is a single strand of DNA or RNA that is complementary to a selected sequence. An antisense RNA oligonucleotide blocks translation of a complementary RNA strand by binding to that RNA. Antisense DNA oligonucleotides can be used to target a specific complementary (coding or non-coding) RNA. Once binding occurs, the DNA / RNA hybrid can be degraded by the enzyme RNase H. In certain embodiments, the antisense oligonucleotide comprises about 10 to about 60 nucleotides, more preferably 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 can be utilized when non-target specific activity is found in the antisense or when an antisense sequence containing one or more mismatches with the target sequence is most preferred for a particular use.

[0189] Antisense oligonucleotides have been shown to be effective and targeted inhibitors of protein synthesis, and therefore can be used to specifically inhibit the protein synthesis of targeted genes.The effectiveness of antisense oligonucleotides for inhibiting protein synthesis has been well established.For example, the synthesis of polygalactauronase and muscarinic type 2 acetylcholine receptor is inhibited by antisense oligonucleotides directed to their respective mRNA sequences (see U.S. Patent Nos. 5,739,119 and 5,759,829). Additionally, examples of antisense inhibition have been shown for the nuclear protein cyclin, the multidrug resistance gene (MDR1), ICAM-1, E-selectin, STK-1, striatal GABAA receptors, and human EGF (see Jaskulski et al., Science, 240:1544-6 (1988); Vasanthakumar et al., Cancer Commun., 1:225-32 (1989); Penis et al., Brain Res Mol Brain Res., 15;57:310-20 (1998); and U.S. Patent Nos. 5,801,154, 5,789,573, 5,718,709, and 5,610,288). Additionally, 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 which are incorporated by reference herein in their entireties for all purposes.

[0190] Methods for generating antisense oligonucleotides are known in the art and can be readily adapted to generate antisense oligonucleotides targeting any polynucleotide sequence. Selection of an antisense oligonucleotide sequence specific for a given target sequence can be determined by analysis of the selected target sequence, as well as secondary structure, T mAntisense 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 in a host cell. Highly preferred target regions of mRNA include the region at or near the AUG translation initiation codon and sequences that are substantially complementary to the 5' region of the mRNA. These secondary structure analyses and target site selection considerations can be performed, for example, using OLIGO primer analysis software v.4 (Molecular Biology Insights) and / or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).

[0191] Ribozymes According to another embodiment of the present invention, the nucleic acid-lipid particle is associated with a ribozyme. Ribozymes are RNA-protein complexes with specific catalytic domains that have 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 catalyze phosphoester transfer reactions with high specificity, often cleaving only one of several phosphates in 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 bind, via specific base-pairing interactions, to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.

[0192] Currently, at least six basic types of naturally occurring enzymatic RNA molecules are known. Each is capable of catalyzing the hydrolysis of RNA phosphodiester bonds in trans (and thus capable of cleaving other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through a target binding portion of the enzymatic nucleic acid, which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, an enzymatic nucleic acid first recognizes a target RNA and then binds to it through complementary base pairing, and upon binding to the correct site, acts enzymatically to cleave the target RNA. Such strategic cleavage of a target RNA abolishes its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to seek another target, and can repeatedly bind to and cleave new targets.

[0193] The enzymatic nucleic acid molecule may be formed, for example, with 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 hammerhead motifs are described, for example, in Rossi et al., Nucleic Acids Res., 20:4559-65 (1992). Examples of hairpin motifs are described, for example, in EP0360257, Hampel et al., Biochemistry, 28:4929-33 (1989), Hampel et al., Nucleic Acids Res., 18:299-304 (1990), and U.S. Patent No. 5,631,359. Examples of hepatitis delta virus motifs are described, for example, in Perrotta et al., Biochemistry, 31:11843-52 (1992). Examples of RNaseP motifs are described, for example, in Guerrier-Takada et al., Cell, 35:849-57 (1983). Examples of Neurospora VS RNA ribozyme motifs are described, for example, in Saville et al., Cell, 61:685-96 (1990), Saville et al., Proc. Natl. Acad. Sci. USA, 88:8826-30 (1991), and Collins et al., Biochemistry, 32:2795-9 (1993). Examples of Group I introns are described, for example, in U.S. Pat. No. 4,987,071. An important feature of the enzymatic nucleic acid molecules used in accordance with the present invention is that they have a specific substrate binding site complementary to one or more of the DNA or RNA regions of the target gene, and that they have a nucleotide sequence within or surrounding the substrate binding site that confers RNA cleavage activity to the molecule. Thus, ribozyme constructs are not necessarily limited to the particular motifs referenced herein, the disclosures of which are incorporated by reference herein in their entireties for all purposes.

[0194] Methods for producing ribozymes that target any polynucleotide sequence are known in the art. Ribozymes can be designed, for example, as described in PCT Publication Nos. WO93 / 23569 and WO94 / 02595, and synthesized for in vitro and / or in vivo testing as described therein. The disclosures of these PCT publications are incorporated herein by reference in their entirety for all purposes.

[0195] Ribozyme activity can be optimized by varying the length of the ribozyme binding arms, or by chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see, e.g., PCT Publication Nos. WO 92 / 07065, WO 93 / 15187, WO 91 / 03162, and WO 94 / 13688, EP 92110298.4, and U.S. Pat. No. 5,334,711, which describe various chemical modifications that can be made to the sugar portion of enzymatic RNA molecules, the disclosures of each of which are incorporated by reference in their entirety for all purposes), modifications that increase their effectiveness within cells, and removal of stem II bases to shorten RNA synthesis time and reduce chemical requirements.

[0196] Immunostimulatory Oligonucleotides The nucleic acid associated with the lipid particles of the present invention can be immunostimulatory, including immunostimulatory oligonucleotides (ISS; single-stranded or double-stranded) that can induce immune response when administered to a subject, which can be a mammal such as a human.ISS can include, for example, a specific palindrome that results in a hairpin secondary structure (see Yamamoto et al., J.Immunol., 148:4072-6 (1992)), or CpG motifs, and other known ISS features (e.g., multiple G domains; see PCT Publication No. WO96 / 11266, the disclosure of which is incorporated herein by reference in its entirety for all purposes).

[0197] Immunostimulatory nucleic acids are considered non-sequence specific if they do not need to specifically bind to and reduce the expression of a target sequence in order to induce an immune response. Thus, certain immunostimulatory nucleic acids may contain sequences that correspond to regions of natural genes or mRNAs, but still be considered non-sequence specific immunostimulatory nucleic acids.

[0198] In one embodiment, the immunostimulatory nucleic acid or oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be unmethylated or methylated. In another embodiment, the immunostimulatory nucleic acid comprises at least one CpG dinucleotide with a methylated cytosine. In one embodiment, the nucleic acid comprises a single CpG dinucleotide, and the cytosine in the CpG dinucleotide is methylated. In an alternative embodiment, the nucleic acid comprises at least two CpG dinucleotides, and at least one cytosine in the CpG dinucleotide is methylated. In a further embodiment, each cytosine in the CpG dinucleotide present in the sequence is methylated. In another embodiment, the nucleic acid comprises a plurality of CpG dinucleotides, and at least one of the CpG dinucleotides comprises a methylated cytosine. Examples of immunostimulatory oligonucleotides suitable for use in the compositions and methods of the 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 each of which are incorporated herein by reference in their entirety for all purposes. In certain embodiments, the oligonucleotides used in the compositions and methods of the invention have a phosphodiester ("PO") or phosphorothioate ("PS") backbone, and / or at least one methylated cytosine residue within a CpG motif.

[0199] mRNA In certain embodiments, the nucleic acid is one or more mRNA molecules (eg, a cocktail of mRNA molecules).

[0200] Modifications to mRNA The mRNA used in the practice of the present invention may contain one, two, or more than two nucleoside modifications. In some embodiments, the modified mRNA exhibits reduced degradation in cells into which the mRNA is introduced, compared to the corresponding unmodified mRNA.

[0201] In some embodiments, modified nucleosides include pyridin-4-one ribonucleosides, 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 ... 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.

[0202] In some embodiments, 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.

[0203] In other embodiments, 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-isopentenyl adenosine, N These include 6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0204] 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 impart stability to the RNA polymer via non-natural phosphorothioate backbone linkage. Phosphorothioate RNA has increased nuclease resistance and subsequently a longer half-life in cellular environment. Phosphorothioate-linked nucleic acids are also expected to reduce innate immune response through relatively weak binding / activation of cellular innate immune molecules.

[0205] In certain embodiments, it is desirable to degrade modified nucleic acids introduced into a cell within the cell, for example when precise timing of protein production is desired. Thus, the present invention provides modified nucleic acids that contain a degradation domain that can act in a directed manner within the cell.

[0206] 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.

[0207] Optional Components of Modified Nucleic Acids In further embodiments, the modified nucleic acid may include other optional components that may be beneficial in some embodiments. These optional components include, but are not limited to, untranslated regions, 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 may be provided, either or both of which may independently include one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the translatable region. Nucleic acids containing Kozak sequences are also provided.

[0208] Additionally, nucleic acids are provided that contain one or more intron nucleotide sequences that can be excised from the nucleic acid.

[0209] Untranslated Regions (UTRs) The untranslated regions (UTRs) of genes are transcribed but not translated. The 5'UTR starts at the transcription initiation site and continues up to, but not including, the initiation codon. The 3'UTR, on the other hand, starts immediately after the stop codon and continues up to the transcription termination signal. There is growing evidence of the regulatory role that UTRs play in the stability and translation of nucleic acid molecules. Regulatory functions of UTRs can be incorporated into the mRNA used in the present invention to increase the stability of the molecule. Specific functions can also be incorporated to ensure that the downregulation of the transcript is controlled when it is misdirected to an undesired organ site.

[0210] 5' Capping The 5' cap structure of mRNA is involved in nuclear export, increases mRNA stability, and binds to mRNA cap-binding protein (CBP), which is involved in mRNA stability and translational competence within the cell through the association of CBP with poly(A)-binding protein to form mature circular mRNA species. The cap also assists in the removal of the 5' proximal intron during mRNA splicing.

[0211] Endogenous mRNA molecules may be capped at the 5' end, resulting in a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the transcribed sense nucleotide at the 5' end of the mRNA molecule. This 5'-guanylic acid cap may then be methylated, resulting in an N7-methyl-guanylic acid residue. The ribose sugar of the 5' end of the mRNA and / or the pre-terminal transcribed nucleotide may also optionally be 2'-O-methylated. 5'-cap removal by hydrolysis and cleavage of the guanylic acid cap structure may target nucleic acid molecules, such as mRNA molecules, for degradation.

[0212] IRES sequence mRNAs containing internal ribosome entry sites (IRES) are also useful in the practice of the present invention. An IRES may function as the only ribosome binding site or as one of multiple ribosome binding sites of an mRNA. An mRNA containing multiple functional ribosome binding sites may code for several peptides or polypeptides that are translated independently by the ribosome ("multicistronic mRNA"). When an mRNA is provided with an IRES, a second translatable region is also optionally provided. Examples of IRES sequences that can be used according to the present invention include, but are not limited to, those derived from picornaviruses (e.g., FMDV), plague virus (CFFV), poliovirus (PV), encephalomyocarditis virus (ECMV), foot and mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (S1V) or cricket paralysis virus (CrPV).

[0213] PolyA tail During RNA processing, long chains of adenine nucleotides (poly-A tails) can be added to polynucleotides such as mRNA molecules to increase stability. Immediately after transcription, the 3' end of the transcript can be cleaved, liberating a 3' hydroxyl. Poly-A polymerase then adds a chain of adenine nucleotides to the RNA. This process, called polyadenylation, adds a poly-A tail that can be 100-250 residues long.

[0214] Typically, the length of the poly-A tail is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (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).

[0215] In this context, the polyA tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer in length 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.

[0216] Creation of mRNA molecules Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989)), as is PCR (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)). Expression libraries are also well known to those skilled in the art. Additional basic documents that disclose 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 incorporated by reference herein in their entireties for all purposes.

[0217] Encoded Polypeptides The mRNA component of the nucleic acid-lipid particles described herein can be used to express a polypeptide of interest. Certain diseases in humans are caused by the absence or impairment of a functional protein in a cell type in which the protein is normally present and active. The functional protein may be completely or partially absent, for example, due to transcriptional inactivity of the encoding gene or due to the presence of a mutation in the encoding gene that renders 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 adrenoleukodystrophy (X-ALD). X-SCID is caused by one or more mutations in the gene encoding the common gamma chain protein, a component of the receptor for several interleukins involved in the development and maturation of B and T cells in the immune system. X-ALD is caused by one or more mutations in the peroxisomal membrane transporter protein gene called ABCD1. Individuals suffering from X-ALD have extremely high levels of long-chain fatty acids in tissues throughout the body, which causes a variety of symptoms that can lead to mental disorders or death.

[0218] Attempts have been made to use gene therapy to treat some diseases caused by the lack or disorder of functional proteins in the cell types in which the proteins are normally present and active.Gene therapy usually involves the introduction of a vector containing a gene that codes for a functional form of the diseased protein into the diseased individual, and the expression of the functional protein to treat the disease.So far, gene therapy has had limited success.Furthermore, certain aspects of using LNP to deliver mRNA have been described, for example, in International Publication Nos. WO2018 / 006052 and WO2015 / 011633.

[0219] Thus, there is a continuing need for improvements to express functional forms of proteins in humans suffering from diseases caused by a complete or partial lack of functional proteins, and for example, there is a need for improved nucleic acid (e.g., mRNA) delivery via methods and compositions that can induce less immune response to therapy. Certain embodiments of the present invention are useful in this context. Thus, in certain embodiments, 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 a lack or reduced levels of functional polypeptides in the human body. In other embodiments, certain compositions and methods of the present invention may be useful for expressing vaccine antigens to treat cancer.

[0220] 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, is generated from a viral genome that lacks at least one structural gene when derived from a positive-stranded virus, and RepRNA can be translated and replicated (hence "self-amplifying") without producing infectious progeny virus. In certain embodiments, RepRNA technology can be used to insert a gene cassette that encodes a desired antigen of interest. For example, alphavirus genomes are divided into two open reading frames (ORFs), the first ORF encoding the proteins of RNA-dependent RNA polymerase (replicase) and the second ORF encoding the structural proteins. In sa-RNA vaccine constructs, the ORF encoding the viral structural proteins can be replaced with any antigen of choice, while the viral replicase remains an integral part of the vaccine, facilitating the intracellular amplification of RNA after immunization.

[0221] Other activators In certain embodiments, the active agent associated with the lipid particles of the present invention may comprise one or more therapeutic proteins, polypeptides, or small organic molecules or compounds.Non-limiting examples of such therapeutically effective agents or drugs include tumor drugs (e.g., chemotherapy drugs, hormonal therapy drugs, immunotherapy drugs, radiotherapy drugs, etc.), lipid-lowering drugs, antiviral drugs, anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, contraceptives, antipyretics, vasodilators, antiangiogenic drugs, cytovascular agents, signal transduction inhibitors, cardiovascular drugs such as antiarrhythmic drugs, hormones, vasoconstrictors, and steroids.These active agents can be administered alone in the lipid particles of the present invention, or can be administered (e.g., co-administered) in combination with the lipid particles of the present invention that contain nucleic acids such as interference RNA or mRNA.

[0222] Non-limiting examples of chemotherapeutic agents include platinum-based drugs (e.g., oxaliplatin, cisplatin, carboplatin, spiroplatin, iproplatin, satraplatin, etc.), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, 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.), topoisomers ... enzyme 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®), sunitinib (Sutent®, SU11248), erlotinib (Tarceva®, OSI-1774), lapatinib (GW572016, GW2016), canertinib (CI1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec®, STI571), dasatinib (BMS-354825), leflunomide (SU101), vandetanib (Zactima™, ZD6474), etc.), pharma- ceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogs thereof, and combinations thereof.

[0223] Examples of conventional hormone therapy agents include, but are not limited to, steroids (eg, dexamethasone), finasteride, aromatase inhibitors, tamoxifen, and goserelin, as well as other gonadotropin-releasing hormone agonists (GnRH).

[0224] Examples of conventional immunotherapeutic agents include 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.), and radioimmunotherapy (e.g., 111 In, 90 Y, or 131 Antibody conjugated to CD20 antibody or IgG1 isoforms include, but are not limited to, anti-CD20 monoclonal antibodies conjugated to CD20 antibody or IgG1.

[0225] Examples of conventional radiotherapeutic agents include, optionally conjugated to antibodies directed against 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 These include, but are not limited to, radionuclides such as Bi.

[0226] Further oncology drugs that can be used according to the invention include alkeran, allopurinol, altretamine, amifostine, anastrozole, araC, arsenic trioxide, bexarotene, biCNU, carmustine, CCNU, celecoxib, cladribine, cyclosporine A, cytosine arabinoside, cytoxan, dexrazoxane, DTIC, estramustine, exemestane, FK506, gemtuzumab-ozogamicin, hydrea, hydroxyurea, idarubicin, interferon, letrozole, leustatin, and the like. These include, but are not limited to, leuprolide, litretinoin, megastrol, L-PAM, mesna, methoxsalen, mithramycin, nitrogen mustard, pamidronate, pegademase, pentostatin, porfimer sodium, prednisone, rituxan, streptozocin, STI-571, taxotere, temozolamide, VM-26, toremifene, tretinoin, ATRA, valrubicin, and velban. Other examples of oncology drugs that can be used according to the present invention are ellipticine and ellipticine analogs or derivatives, epothilones, intracellular kinase inhibitors, and camptothecin.

[0227] 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.

[0228] Examples of antiviral agents include abacavir, aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripla, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fixed dose combinations, fomivirsen, fosamprenavir, foscarnet, phosphonet, fusion inhibitors, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors, type III interferons (e.g., IFN- IFN-lambda molecules such as IFN-lambda 1, IFN-lambda 2, and IFN-lambda 3), type II interferons (e.g., IFN-gamma), type I interferons (e.g., IFN-alpha such as PEGylated IFN-alpha, IFN-beta, IFN-kappa, IFN-delta, IFN-epsilon, IFN-tau, IFN-omega, and IFN-zeta), interferons, lamibcin, lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, synergistic enhancers enhancer), tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, zidovudine, pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogs thereof, and mixtures thereof.

[0229] lipid particles The lipid particles of the present invention typically include an active or therapeutic agent, a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits particle aggregation. In some embodiments, the active or therapeutic agent is fully 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, for example by nucleases or proteases, in aqueous solution. In other embodiments, the lipid particles described herein are substantially non-toxic to mammals, such as humans. The lipid particles of the present invention typically have an average diameter of about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm.

[0230] In a preferred embodiment, the lipid particles of the present invention are serum-stable nucleic acid-lipid particles (LNPs) that contain one or more nucleic acid molecules, such as interfering RNA (e.g., siRNA, aiRNA, and / or miRNA) or mRNA, cationic lipids (e.g., cationic lipids of formula I, II, and / or III), non-cationic lipids (e.g., cholesterol alone or a mixture of one or more phospholipids and cholesterol), and conjugated lipids that inhibit particle aggregation (e.g., one or more PEG-lipid conjugates). LNPs can contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 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, as well as PCT Publication No. WO 96 / 40964, the disclosures of each of which are incorporated by reference herein in their entirety for all purposes.

[0231] Cationic lipids In the lipid nanoparticles of the present invention, the cationic lipid may be selected from compounds of formula (I): [ka] (In the formula, R 1 is C2~C 30 is a hydrocarbyl; R 2 is C2~C 30 is a hydrocarbyl; R 3 is C2~C 30 is a hydrocarbyl; X is a divalent C2-C8 alkyl; R 4 is NR a R b and; R a and R b are each independently selected from the group consisting of methyl, ethyl, propyl, cyclopropyl, and butyl, wherein methyl, ethyl, propyl, cyclopropyl, and butyl are optionally substituted with hydroxy; or R a and R b together with the nitrogen to which they are attached form an aziridine ring, azetidine ring, proline ring, piperidine ring, piperazine ring, or morpholine ring, which is optionally substituted with hydroxyl or C1-C6 alkyl optionally substituted with hydroxyl.

[0232] In one embodiment, R 1 is C2~C 20 It is a hydrocarbyl.

[0233] In one embodiment, R 1 is C2~C 15 It is a hydrocarbyl.

[0234] In one embodiment, R 1 is C2~C 10 It is a hydrocarbyl.

[0235] In one embodiment, R 1 is C5~C 20 It is a hydrocarbyl.

[0236] In one embodiment, R1 (C2~C 20 ) Alkyl, (C2-C 20 ) alkenyl, or (C2-C 20 ) alkynyl.

[0237] In one embodiment, R 1 (C8~C 20 ) alkyl.

[0238] In one embodiment, R 1 (C8~C 20 ) alkenyl.

[0239] In one embodiment, R 1 (C8~C 20 ) alkynyl.

[0240] In one embodiment, R 1 has only one double bond (C8-C 20 ) alkenyl.

[0241] In one embodiment, R 1 is (Z)-4-decen-1-yl, 1-nonyl, 3-undecyl, 1-decyl, 6(Z),15(Z)-henicosandiene-11-yl, 3-hexen-1-yl, 9(Z)-octadecen-1-yl, or 2-butyloct-1-yl, 4-(1-methylethenyl)cyclohexen-1-ylmethyl.

[0242] In one embodiment, R 2 is C2~C 20 It is a hydrocarbyl.

[0243] In one embodiment, R 2 is C2~C 15 It is a hydrocarbyl.

[0244] In one embodiment, R 2 is C2~C 10 It is a hydrocarbyl.

[0245] In one embodiment, R 2 is C5~C 20 It is a hydrocarbyl.

[0246] In one embodiment, R 2 (C2~C 20 ) Alkyl, (C2-C 20 ) alkenyl, or (C2-C 20 ) alkynyl.

[0247] In one embodiment, R 2 (C8~C 20 ) alkyl.

[0248] In one embodiment, R 2 (C8~C 20 ) alkenyl.

[0249] In one embodiment, R 2 (C8~C 20 ) alkynyl.

[0250] In one embodiment, R 2 has only one double bond (C8-C 20 ) alkenyl.

[0251] In one embodiment, R 2 is (Z)-4-decen-1-yl, 1-nonyl, 3-undecyl, 1-decyl, 6(Z),15(Z)-henicosanedien-11-yl, 3-hexen-1-yl, 9(Z)-octadecen-1-yl, or 2-butyloct-1-yl, 4-(1-methylethenyl)cyclohexen-1-ylmethyl.

[0252] In one embodiment, R 3 is C2~C 20 It is a hydrocarbyl.

[0253] In one embodiment, R 3 is C2~C 15 It is a hydrocarbyl.

[0254] In one embodiment, R 3 is C2~C 10 It is a hydrocarbyl.

[0255] In one embodiment, R 3 is C5~C 20 It is a hydrocarbyl.

[0256] In one embodiment, R 3 (C2~C 20 ) Alkyl, (C2-C 20 ) alkenyl, or (C2-C 20 ) alkynyl.

[0257] In one embodiment, R 3 (C8~C 20 ) alkyl.

[0258] In one embodiment, R 3 (C8~C 20 ) alkenyl.

[0259] In one embodiment, R 3 (C8~C 20 ) alkynyl.

[0260] In one embodiment, R 3 has only one double bond (C8-C 20 ) alkenyl.

[0261] In one embodiment, R 3 is (Z)-4-decen-1-yl, 1-nonyl, 3-undecyl, 1-decyl, 6(Z),15(Z)-henicosanedien-11-yl, 3-hexen-1-yl, 9(Z)-octadecen-1-yl, adamantly-1-ylmethyl, 2-butyloct-1-yl, (Z)-2-((Z)-dec-4-en-1-yl)dodec-6-en-1-yl, or 4-(prop-1-en-2-yl)cyclohex-1-en-1-yl)methyl.

[0262] In one embodiment, X is a divalent C2-C6 alkyl.

[0263] In one embodiment, X is a divalent C3-C5 alkyl.

[0264] In one embodiment, X is -CH2CH2CH2-.

[0265] In one embodiment, X is -CH2CH2CH2CH2-.

[0266] In one embodiment, X is -CH2CH2CH2CH2CH2-.

[0267] In one embodiment, R a and R b are each independently selected from the group consisting of methyl, ethyl, propyl, cyclopropyl, and butyl, wherein methyl, ethyl, propyl, cyclopropyl, and butyl are optionally substituted with hydroxy.

[0268] In one embodiment, R a and R b together with the nitrogen to which they are attached form an aziridine ring, azetidine ring, proline ring, piperidine ring, piperazine ring, or morpholine ring, which is optionally substituted with hydroxyl or C1-C6 alkyl optionally substituted with hydroxyl.

[0269] In one embodiment, R a and R b are each independently selected from the group consisting of methyl and ethyl.

[0270] In one embodiment, R 4 is dimethylamino.

[0271] Non-cationic lipids The non-cationic lipids used in the lipid particles (e.g., LNPs) of the present invention can be any of a variety of neutral uncharged, zwitterionic, or anionic lipids capable of forming a stable complex.

[0272] Non-limiting examples of non-cationic lipids include phospholipids, such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine ...DPPC), palmitoyloleoyl-phosphatidylcholine (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phos Phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably C 10 ~C 24 Acyl groups derived from fatty acids having carbon chains, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0273] 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.

[0274] In some embodiments, the non-cationic lipid present in the lipid particle (e.g., LNP) comprises or consists of cholesterol or its derivatives, e.g., lipid particle formulations that do not contain phospholipids.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., lipid particle formulations that do not contain cholesterol.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 derivatives.

[0275] Other examples of non-cationic lipids suitable for use in the present invention include phosphorus-free lipids such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, sphingomyelin, and the like.

[0276] In some embodiments, the non-cationic lipid may constitute about 13 mol% to about 49.5 mol%, about 20 mol% to about 45 mol%, about 25 mol% to about 45 mol%, about 30 mol% to about 45 mol%, about 35 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 40 mol%, or about 30 mol% to about 40 mol% of the total lipid present in the particle.

[0277] In certain embodiments, the cholesterol present in the phospholipid-free lipid particle comprises 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 lipid present in the particle. As a non-limiting example, the phospholipid-free lipid particle may comprise about 37 mol% cholesterol of the total lipid present in the particle.

[0278] In certain other embodiments, the cholesterol present in a lipid particle containing a mixture of phospholipids and cholesterol comprises 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 lipid present in the particle. As a non-limiting example, a lipid particle comprising a mixture of phospholipids and cholesterol may contain about 34 mol% cholesterol of the total lipid present in the particle.

[0279] In further embodiments, the cholesterol present in a lipid particle containing a mixture of phospholipids and cholesterol comprises 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 lipid present in the particle. As a non-limiting example, a lipid particle comprising a mixture of phospholipids and cholesterol may contain about 20 mol% cholesterol of the total lipid present in the particle.

[0280] In embodiments in which the lipid particle contains a mixture of phospholipids and cholesterol or cholesterol derivatives, the mixture may comprise up to about 40, 45, 50, 55, or 60 mol% of the total lipid present in the particle. In certain cases, the phospholipid component in the mixture may comprise 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 lipid present in the particle. As a non-limiting example, a lipid particle comprising a mixture of phospholipids and cholesterol may contain about 7 mol% of phospholipids such as DPPC or DSPC of the total lipid present in the particle (e.g., in a mixture with about 34 mol% cholesterol). In certain other cases, the phospholipid component in the mixture may comprise 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 lipid present in the particle. As another non-limiting example, a lipid particle comprising a mixture of phospholipids and cholesterol may contain about 20 mol % phospholipid such as DPPC or DSPC of the total lipid present in the particle (e.g., in a mixture with about 20 mol % cholesterol).

[0281] Lipid conjugates In addition to cationic and non-cationic lipids, the lipid particles (e.g., LNPs) of the present invention comprise lipid conjugates. Conjugated lipids are useful in inhibiting particle aggregation. Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, ATTA-lipid conjugates, cationic polymer-lipid conjugates (CPLs), and mixtures thereof. In certain embodiments, the particles comprise either PEG-lipid conjugates or ATTA-lipid conjugates together with CPLs.

[0282] In a preferred embodiment, the lipid conjugate is a PEG-lipid.Examples of PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyl (PEG-DAA), as described, for example, in PCT Publication No. 05 / 026372, PEG coupled to diacylglycerol (PEG-DAG), as described, for example, in US Patent Publication Nos. 20030077829 and 2005008689, PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramide, as described, for example, in US Patent No. 5,885,613, PEG conjugated to cholesterol or its derivatives, and mixtures thereof.The disclosures of these patent documents are incorporated herein by reference in their entirety for all purposes.Additional PEG-lipids include, but are not limited to, PEG-C-DOMG, 2KPEG-DMG, and mixtures thereof.

[0283] PEG is a linear water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEG is classified according to its molecular weight, for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. PEG is commercially available from Sigma Chemical Co. and other companies, including, for example, the following: monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH2), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), and monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM). Other PEGs (e.g., mPEG (20KDa) amines), such as those described in U.S. Patent Nos. 6,774,180 and 7,053,150, are also useful for preparing the PEG-lipid conjugates of the present invention. The disclosures of these patents are incorporated by reference in their entirety for all purposes. In addition, monomethoxypolyethylene glycol acetic acid (MePEG-CH2COOH) is particularly useful for preparing PEG-lipid conjugates, including, for example, PEG-DAA conjugates.

[0284] The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from 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 preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0285] In certain cases, PEG may be optionally substituted with an alkyl group, an alkoxy group, an acyl group, or an aryl group. PEG may be directly conjugated to lipid or may be linked to lipid via a linker moiety. Any linker moiety suitable for coupling PEG to lipid may be used, including, for example, ester-free linker moieties and ester-containing linker moieties. 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 non-ester containing 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 (-SS-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, disulfide, and combinations thereof (e.g., linkers containing both carbamate and amide linker moieties). In a preferred embodiment, a carbamate linker is used to couple the PEG to the lipid.

[0286] In other embodiments, an ester-containing linker moiety is used to couple the 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.

[0287] Additional PEG-lipid conjugates suitable for use in the present invention include, but are not limited to, those having the formula: [ka] [In the formula, A is (C1-C6)alkyl, (C3-C8)cycloalkyl, (C3-C8)cycloalkyl(C1-C6)alkyl, (C1-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (C1-C6)alkanoyl, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, or (C2-C6)alkanoyloxy, where any of (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 included. yloxy is substituted with one or more anion precursor groups, and wherein any of the (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; B is a polyethylene glycol chain having a molecular weight of about 550 daltons to about 10,000 daltons; C is -LR a (L is a direct bond, -C(O)O-, -C(O)NR b -, -NR b -, -C(O)-, -NR b C(O)O-, -NR b C(O)NR b selected from the group consisting of -, -SS-, -O-, -(O)CCH2CH2C(O)-, and -NHC(O)CH2CH2C(O)NH-; R a is a branched (C 10 ~C 50) alkyl or branched (C 10 ~C 50 ) alkenyl, where branched (C 10 ~C 50 ) alkyl or branched (C 10 ~C 50 ) one or more carbon atoms of the alkenyl is replaced with -O-; R b are each independently H or (C1-C6) alkyl), or a salt thereof.

[0288] The conjugated lipid may comprise, for example, a PEG-lipid comprising a compound of the formula A-PEG-diacylglycerol (DAG), A-PEG-dialkyloxypropyl (DAA), A-PEG-phospholipid, A-PEG-ceramide (Cer), or a mixture thereof, where A is selected from the group consisting of (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 of (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 of (C1-C6)alkyl, (C3-C8)cycloalkyl, (C3-C8)cycloalkyl(C1-C6)alkyl, (C1-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (C2-C6)alkoxy ... The (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-dilauryloxypropyl (C12), A-PEG-dimyristyloxypropyl (C14), A-PEG-dipalmityloxypropyl (C16), or A-PEG-distearyloxypropyl (C18), or mixtures thereof.

[0289] 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 are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. 10 ~C 20 Phosphatidylethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths in the range of 0.1 to 100 are preferred. Phosphatidylethanolamines with 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).

[0290] The term "ATTR" or "polyamide" refers, without limitation, to compounds described in U.S. Patent Nos. 6,320,017 and 6,586,559, the disclosures of which are incorporated by reference in their entirety for all purposes. These compounds include those having the formula: [ka] wherein R is a member selected from the group consisting of hydrogen, alkyl, and acyl; R 1 is a member selected from the group consisting of hydrogen and alkyl; or optionally, R and R 1 and the nitrogen to which they are attached form an azide moiety; R 2 is a member of the group selected from hydrogen, optionally substituted alkyl, optionally substituted aryl, and an amino acid side chain; R 3 is hydrogen, halogen, hydroxy, alkoxy, mercapto, hydrazino, amino and NR 4 R 5 (In the formula, R 4 and R5 are independently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p is 1 to 4; and q is 0 or 1. It will be apparent to one of ordinary skill in the art that other polyamides can be used in the compounds of the present invention.

[0291] The term "diacylglycerol" refers to the two fatty acyl chains R 1 and R 2 which both have 2-30 carbons attached independently to the 1- and 2-positions of glycerol by ester bonds. The acyl groups may be saturated or have various degrees of unsaturation. Suitable acyl groups include lauryl (C 12 ), myristyl (C 14 ), Palmityl (C 16 ), Stearyl (C 18 ), and Eicosil (C 20 In a preferred embodiment, R 1 and R 2 are the same, i.e., R 1 and R 2 are both myristyl (i.e. dimyristyl), R 1 and R 2 are all stearyl (i.e., distearyl), etc. Diacylglycerols have the following general formula: [ka] has.

[0292] The term "dialkyloxypropyl" refers to two alkyl chains, R 1 and R 2 which both independently have 2 to 30 carbons. The alkyl groups may be saturated or have varying degrees of unsaturation. Dialkyloxypropyl refers to compounds having the following general formula: [ka] has.

[0293] In a preferred embodiment, the PEG-lipid has the following formula: [ka] wherein R 1 and R 2 are independently selected and are long chain alkyl groups having from about 10 to about 22 carbon atoms; PEG is polyethylene glycol; and L is an ester-free or ester-containing linker moiety as described above. The long chain alkyl groups can be saturated or unsaturated. Suitable alkyl groups include lauryl (C 12 ), myristyl (C 14 ), Palmityl (C 16 ), Stearyl (C 18 ), and Eicosil (C 20 In a preferred embodiment, R 1 and R 2 are the same, i.e., R 1 and R 2 are both myristyl (i.e. dimyristyl), R 1 and R 2 are all stearyl (i.e. distearyl), etc.

[0294] In the above formula VII, PEG has an average molecular weight ranging from 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 be optionally substituted with alkyl, alkoxy, acyl, or aryl. In certain embodiments, the terminal hydroxyl group is substituted with a methoxy group or a methyl group.

[0295] In a preferred embodiment, "L" is an ester-free linker moiety. Suitable ester-free linkers include, but are not limited to, amide linker moieties, amino linker moieties, carbonyl linker moieties, carbamate linker moieties, urea linker moieties, ether linker moieties, disulfide linker moieties, succinamidyl linker moieties, and combinations thereof. In a preferred embodiment, the ester-free linker moiety is a carbamate linker moiety (i.e., a PEG-C-DAA conjugate). In another preferred embodiment, the ester-free linker moiety is an amide linker moiety (i.e., a PEG-A-DAA conjugate). In yet another preferred embodiment, the ester-free linker moiety is a succinamidyl linker moiety (i.e., a PEG-S-DAA conjugate).

[0296] In certain embodiments, the PEG-lipid conjugate is selected from the following: [ka] In one embodiment, n is selected so that the resulting polymer chain has a molecular weight of about 2000.

[0297] PEG-DAA conjugates are synthesized using standard techniques and reagents known to those skilled in the art. It will be recognized that PEG-DAA conjugates contain various amide, amine, ether, thio, carbamate, and urea bonds. Those skilled in the art will recognize that methods and reagents for forming these bonds are well known and readily available. See, for example, March, ADVANCED ORGANIC CHEMISTRY (Wiley 1992), Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989), and Furniss, 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 PEG-DAA conjugates. 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).

[0298] Preferably, the PEG-DAA conjugate is dilauryloxypropyl (C 12 )-PEG conjugate, dimyristyloxypropyl (C 14 )-PEG conjugate, dipalmityloxypropyl (C 16 )-PEG conjugates, or distearyloxypropyl (C 18 )-PEG conjugates. One of skill in the art will readily appreciate that other dialkyloxypropyls can be used in the PEG-DAA conjugates of the present invention.

[0299] In addition to the above, it will be readily apparent to one 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.

[0300] The charge of the polycationic moiety can be either distributed over the entire particle portion, or alternatively, can be a charge density of different concentration in certain regions of the particle portion, e.g., a charge spike. When the charge density is distributed on the particle, the charge density can be distributed uniformly or non-uniformly. All variations in the charge distribution of the polycationic moiety are encompassed by the present invention.

[0301] The lipid "A" and the 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 to covalently link "A" and "W". Suitable bonds include, but are not limited to, amide bonds, amine bonds, carboxyl bonds, carbonate bonds, carbamate bonds, ester bonds, and hydrazone bonds. It will be clear to those skilled in the art that "A" and "W" must have complementary functional groups to effect the linkage. The reaction of these two groups, one on the lipid and the other on the polymer, will result in the desired linkage. For example, if the lipid is a diacylglycerol and the terminal hydroxyl is activated, for example with NHS and DCC to form an active ester, and then this lipid is reacted with a polymer containing an amino group, such as a polyamide (see, for example, U.S. Patent Nos. 6,320,017 and 6,586,559, the disclosures of which are incorporated by reference in their entirety for all purposes), an amide bond will be formed between the two groups.

[0302] In certain cases, the polycationic moiety may be bound to a ligand, such as a targeting ligand or a chelating moiety for complexing calcium. Preferably, the cationic moiety maintains a positive charge after the ligand is bound. In certain cases, the bound ligand has a positive charge. Suitable ligands include, but are not limited to, compounds or devices with reactive functional groups, including lipids, amphiphilic lipids, carrier compounds, biocompatible compounds, biomaterials, biopolymers, medical devices, analytically detectable compounds, therapeutically active compounds, enzymes, peptides, proteins, antibodies, immunostimulants, radiolabels, fluorogenic substances, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, other targeting moieties, or toxins.

[0303] 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 lipid present in the particle.

[0304] One of skill in the art will understand that the concentration of lipid conjugate can be varied depending on the lipid conjugate used and the rate at which the nucleic acid-lipid particle becomes fusogenic.

[0305] By controlling the composition and concentration of lipid conjugate, the rate at which lipid conjugate is exchanged with the outside of nucleic acid-lipid particle and then the rate at which nucleic acid-lipid particle becomes fusogenic can be controlled.For example, when using PEG-phosphatidylethanolamine conjugate or PEG-ceramide conjugate as lipid conjugate, the rate at which nucleic acid-lipid particle becomes fusogenic can be changed, for example, by changing the concentration of lipid conjugate, by changing the molecular weight of PEG, or by changing the chain length and saturation of acyl chain group on phosphatidylethanolamine or ceramide.In addition, other variables, including, for example, pH, temperature, ionic strength, etc., can be used to change and / or control the rate at which nucleic acid-lipid particle becomes fusogenic.Other methods that can be used to control the rate at which nucleic acid-lipid particle becomes fusogenic will be clear to those skilled in the art upon reading this disclosure.

[0306] Preparation of lipid particles The lipid particles of the present invention, e.g., LNPs, in which an active or therapeutic agent, such as a nucleic acid molecule, is encapsulated in the lipid bilayer and protected from degradation, can be formed by any method known in the art, including, but not limited to, continuous mixing methods or direct dilution processes.

[0307] 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), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (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-dielideyl-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.

[0308] In certain embodiments, the invention provides LNPs produced by a continuous mixing method, e.g., a process that includes providing an aqueous solution containing a nucleic acid, such as an interfering RNA or mRNA, in a first reservoir, providing an organic lipid solution in a second reservoir, and mixing the aqueous solution with the organic lipid solution such that the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce liposomes that encapsulate the nucleic acid (e.g., the interfering RNA or mRNA). This process and an apparatus for carrying out this process are described in detail in U.S. Patent Application Publication No. 20040142025, the disclosure of which is incorporated by reference herein in its entirety for all purposes.

[0309] The operation of successively introducing lipid and buffer solutions into a mixing environment, such as in a mixing chamber, results in the successive dilution of the lipid solution with the buffer solution, which produces liposomes substantially instantly upon mixing.As used herein, the phrase "successively diluting the lipid solution with the buffer solution" (and variations) generally means that the lipid solution is diluted sufficiently quickly and with sufficient force to produce vesicles in the hydration process.By mixing an aqueous solution containing nucleic acid with an organic lipid solution, the organic lipid solution undergoes a successive stepwise dilution in the presence of a buffer solution (i.e., an aqueous solution) to produce nucleic acid-lipid particles.

[0310] LNPs formed using continuous mixing methods 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 size.

[0311] In another embodiment, the present invention provides LNPs produced by a direct dilution process, which includes forming a liposome solution and immediately and directly introducing the liposome solution into a collection vessel containing a controlled amount of dilution buffer. In a preferred embodiment, the collection vessel includes one or more components configured to agitate the contents of the collection vessel to facilitate dilution. In one embodiment, the amount of dilution buffer present in the collection vessel is substantially equal to the volume of the liposome solution introduced therein. As a non-limiting example, a liposome solution in 45% ethanol will advantageously result in smaller particles when introduced into a collection vessel containing an equal volume of dilution buffer.

[0312] 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 the second mixing region. In this embodiment, the liposome solution formed in the first mixing region is immediately mixed directly with the dilution buffer in the second mixing region. In a preferred aspect, the second mixing region includes a T-connector arranged such that the flow of the liposome solution and the dilution buffer meet as 180° opposing flows, although connectors that result in shallower angles, e.g., from about 27° to about 180°, can be used. A pump mechanism delivers a controllable buffer flow to the second mixing region. In one aspect, the flow rate of the dilution buffer provided 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 therefore also the concentration of the liposome solution in the buffer throughout the second mixing process. Such control of the dilution buffer flow rate advantageously allows for the formation of small particle sizes at reduced concentrations.

[0313] These processes and equipment 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 by reference herein in its entirety for all purposes.

[0314] LNPs formed using the direct dilution process 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 size.

[0315] If necessary, 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.

[0316] 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. Pat. No. 4,737,323, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Sonicating the particle suspension, either by bath sonication or probe sonication, results in a gradual reduction in size to particles less than about 50 nm in size. 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 is observed, typically about 60 to about 80 nm. In both methods, the particle size distribution can be monitored by conventional laser light particle size discrimination or QELS.

[0317] Extrusion of particles through fine-pored polycarbonate or asymmetric ceramic membranes is also an effective method for reducing particle size to a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired particle size distribution is obtained. The particles can be extruded through successive membranes with smaller pores to gradually reduce the size.

[0318] In some embodiments, the nucleic acids in the LNPs are pre-concentrated, for example, as described in U.S. Patent Application No. 09 / 744,103, the disclosure of which is incorporated by reference in its entirety for all purposes.

[0319] In another embodiment, the method further comprises adding a non-lipid polycation useful for lipofection of cells using the composition 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.

[0320] 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, about 0.02 to about 0.1, about 0.03 to about 0.1, or about 0.01 to about 0.08. The ratio of the starting materials also falls within this range. In other embodiments, the LNP preparation uses about 400 μg of nucleic acid per 10 mg of total lipid, or a nucleic acid to lipid mass ratio of about 0.01 to about 0.08, more preferably about 0.04, corresponding to 1.25 mg of total lipid per 50 μg of nucleic acid. In other preferred embodiments, the particles have a nucleic acid:lipid mass ratio of about 0.08.

[0321] In other embodiments, the lipid to nucleic acid ratio (mass / mass ratio) in the formed LNP is 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), about 4 (4:1), or the like. ) to about 50 (50:1), about 5 (5:1) to about 50 (50:1), about 1 (1:1) to about 25 (25:1), about 2 (2:1) to about 25 (25:1), about 3 (3:1) to about 25 (25:1), about 4 (4:1) to about 25 (25:1), about 5 (5:1) to about 25 (25:1), about 5 (5:1) to about 20 (20: 1), about 5 (5:1) to about 15 (15:1), 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 the starting materials also typically falls within this range.

[0322] As mentioned above, the conjugate lipid may further include CPL. Various general methods for making LNP-CPL (CPL-containing LNP) are discussed herein. Two general techniques include "post-insertion" technique, i.e., inserting CPL into, for example, preformed LNP, and "standard" technique, in which CPL is included in the lipid mixture during, for example, the LNP formation process. The post-insertion technique produces LNPs with CPL mainly on the outer surface of the LNP bilayer membrane, while the standard technique results in LNPs with CPL on both the inner and outer surfaces. This method is particularly useful for vesicles made from phospholipids (which may contain cholesterol), and also for vesicles containing PEG-lipids (e.g., PEG-DAA and PEG-DAG). Methods for making LNP-CPLs 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 incorporated by reference in their entireties for all purposes.

[0323] Other methods for making LNPs can be found, for example, in U.S. Pat. No. 9,005,654 and PCT Publication No. 2007 / 012191, the disclosures of which are incorporated by reference herein in their entireties for all purposes.

[0324] kit The present invention also provides lipid particles (e.g., LNPs) in kit form. The kit may include compartmentalized containers to hold various components of the lipid particle (e.g., an active agent or therapeutic agent, such as a nucleic acid, and individual lipid components of the particle). In some embodiments, the kit may further include an endosomal membrane destabilizing agent (e.g., calcium ions). The kit typically includes the lipid particle composition of the present invention, preferably in dehydrated form, together with instructions for its rehydration and administration.

[0325] As described herein, lipid particles (e.g., LNPs) of the present invention can be adapted to preferentially target specific tissues, organs, or tumors of interest.In certain cases, preferential targeting of lipid particles, such as LNPs, can be achieved by controlling the composition of the particle itself.For example, as described in Example 11, it has been found that 1:57 PEG-cDSA LNP formulation can be used to preferentially target tumors outside the liver, while 1:57 PEG-cDMA LNP formulation can be used to preferentially target the liver (including liver tumors).

[0326] In certain other cases, it may be desirable to have targeting moieties attached to the surface of the lipid particle to further enhance targeting of the particle. Methods for attaching targeting moieties (e.g., antibodies, proteins, etc.) to lipids (such as those used in the particles of the present invention) are known to those of skill in the art.

[0327] Administration of lipid particles The lipid particles (e.g., LNPs) of the present invention are useful for introducing active or therapeutic agents (e.g., nucleic acids such as interfering RNA or mRNA) into cells after they are formed.Thus, the present invention also provides a method for introducing active or therapeutic agents, such as nucleic acids (e.g., interfering RNA or mRNA), into cells.This method is carried out in vitro or in vivo by first forming particles as described above, and then contacting the particles with cells for a period of time sufficient to cause delivery of the active or therapeutic agent to the cells.

[0328] The lipid particles (e.g., LNPs) of the present invention can be adsorbed to almost any cell type that is mixed or contacted with them. Once adsorbed, the particles can either be endocytosed by a portion of the cells, exchange lipids with the cell membrane, or fuse with the cells. The introduction or incorporation of the active agent or therapeutic agent (e.g., nucleic acid) portion of the particle can occur via any one of these routes. In particular, when fusion occurs, the particle membrane is incorporated into the cell membrane and the particle contents mix with intracellular fluid.

[0329] The lipid particles (e.g., LNPs) of the present invention can be administered either alone or in admixture with a pharma- ceutically acceptable carrier (e.g., saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practice. Generally, buffered saline (e.g., 135-150 mM NaCl) will be used as the pharma- ceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoproteins, globulins, and the like. 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, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The phrase "pharmacologically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans.

[0330] The pharma- ceutically acceptable carrier is generally added after the lipid particles are formed. Thus, after the particles are formed, they can be diluted in a pharma- ceutically acceptable carrier, such as physiological buffered saline.

[0331] The concentration of particles in a pharmaceutical formulation can vary greatly, i.e., less than about 0.05% by weight, usually from 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 by fluid volume, viscosity, etc., according to the particular mode of administration selected. For example, the concentration can be increased to reduce the fluid load associated with the treatment. This may be particularly desirable for patients with congestive heart failure or severe hypertension associated with atherosclerosis. Alternatively, particles composed of irritating lipids may be diluted to low concentrations to reduce inflammation at the site of administration.

[0332] The pharmaceutical composition of the present invention may be sterilized by conventional well-known sterilization techniques. The aqueous solution may be packaged for use or filtered under aseptic conditions and lyophilized, and the lyophilized preparation may be mixed with a sterile aqueous solution before administration. The composition may contain pharma- ceutically acceptable auxiliary substances such as pH adjusting and buffering agents, osmolality adjusting agents, etc., required to approximate physiological conditions, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride. In addition, the particle suspension may contain lipid protective agents that protect lipids from damage caused by free radicals and lipid peroxidation during storage. Lipophilic free radical quenchers such as alpha-tocopherol and water-soluble iron-specific chelators such as ferrioxamine are suitable.

[0333] In vivo administration Systemic delivery for in vivo therapy, for example, delivery of therapeutic nucleic acids to distant target cells via a body system such as the circulation, has been achieved using nucleic acid-lipid particles such as those described in PCT Publication Nos. WO05 / 007196, WO05 / 121348, WO05 / 120152, and WO04 / 002453, the disclosures of which are incorporated herein by reference in their entirety for all purposes.The present invention also provides fully encapsulated lipid particles that protect nucleic acids from nuclease degradation in serum, are non-immunogenic, and have a small size suitable for repeated dosing.

[0334] For in vivo administration, 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. Administration can be by single or divided doses. The pharmaceutical composition can be administered parenterally, i.e., intraarticularly, 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). Additional methods of administering lipid-based therapeutic agents are described, for example, 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 disease site or by injection at a site distal to the disease site (see, e.g., Culver, HUMAN GENE THERAPY, Mary Ann Liebert, Inc., Publishers, New York. pp. 70-71 (1994)). The disclosures of the aforementioned references are incorporated herein by reference in their entirety for all purposes.

[0335] The compositions of the present invention can be made into aerosol formulations (i.e., they can be "nebulized"), either alone or in combination with other suitable components, for administration via inhalation (e.g., intranasally or intratracheally) (see Brigham et al., Am. J. Sci., 298:278 (1989)). The aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0336] In certain embodiments, pharmaceutical compositions can be delivered by intranasal spray, inhalation, and / or other aerosol delivery vehicles. Methods for delivering nucleic acid compositions directly to the lungs by intranasal aerosol spray are described, for example, in U.S. Patent Nos. 5,756,353 and 5,804,212. Similarly, drug delivery using intranasal microparticle resins and lysophosphatidyl-glycerol compounds (U.S. Patent No. 5,725,871) is also well known in the pharmaceutical field. Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is ​​described in U.S. Patent No. 5,780,045. The disclosures of the aforementioned patents are incorporated herein by reference in their entirety for all purposes.

[0337] Formulations suitable for parenteral administration, such as by intraarticular (into a joint), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions are preferably administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally.

[0338] Generally, when administered intravenously, the lipid particle formulation is formulated with a suitable pharmaceutical carrier. Many pharma- ceutically 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). A variety of aqueous carriers can be used, such as water, buffered water, 0.4% saline, 0.3% glycine, and the like, which may contain glycoproteins for enhanced stability, such as albumin, lipoproteins, globulins, and the like. Generally, buffered saline (135-150 mM NaCl) is used as a pharma- ceutically acceptable carrier, although other suitable carriers would suffice. These compositions can be sterilized by conventional liposome sterilization techniques, such as filtration. The compositions may contain pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, osmolality adjusting agents, wetting agents, etc., required to approximate physiological conditions, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. These compositions may be sterilized using the techniques described above, or alternatively, may be produced under sterile conditions. The resulting aqueous solutions may be packaged for use, or may be filtered and lyophilized under aseptic conditions, and the lyophilized preparation may be mixed with a sterile aqueous solution prior to administration.

[0339] 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, oblates, etc. (see, for example, U.S. Patent Nos. 5,641,515, 5,580,579, and 5,792,451, the disclosures of which are incorporated herein by reference in their entirety for all purposes). These oral dosage forms can also contain the following: binders, gelatin; excipients, lubricants, and / or flavoring agents. When the unit dosage form is a capsule, the dosage form can contain a liquid carrier in addition to the above-mentioned materials. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. Of course, any material used in preparing any unit dosage form should be pharma- ceutical pure and substantially non-toxic in the amounts employed.

[0340] Typically, these oral formulations may contain at least about 0.1% or more lipid particles, although the percentage of particles may of course vary and may conveniently be from about 1% or 2% to about 60% or 70% or more of the total weight or volume of the formulation. Naturally, the amount of particles in each therapeutically useful composition may be prepared in such a way 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 of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimes may be desirable.

[0341] Formulations suitable for oral administration may consist of the following: (a) an effective amount of a packaged therapeutic agent, such as a nucleic acid (e.g., interfering RNA or mRNA), suspended in a liquid solution, e.g., a diluent such as water, saline, or PEG 400; (b) a capsule, sachet, or tablet containing a predetermined amount of a therapeutic agent, such as a nucleic acid (e.g., interfering RNA or mRNA), as a liquid, solid, granule, or gelatin, respectively; (c) a suspension in a suitable liquid; and (d) a suitable emulsion. Tablet forms may contain one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavorings, dyes, disintegrants, and pharma- ceutically compatible carriers. The lozenge form may include a pastille containing the therapeutic agent in an inert base such as a flavoring, e.g., a therapeutic agent such as a nucleic acid (e.g., interfering RNA or mRNA) in sucrose, and a carrier known in the art in addition to the therapeutic agent, such as gelatin and glycerin, or a sucrose and gum arabic emulsion, gel.

[0342] In another example of their use, the lipid particles can be incorporated into a wide variety of topical dosage forms. For example, suspensions containing nucleic acid-lipid particles such as LNPs can be formulated and administered as gels, oils, emulsions, topical creams, pastes, ointments, lotions, foams, mousses, and the like.

[0343] When preparing pharmaceutical preparations of the lipid particles of the present invention, it is preferable to use large amounts of particles that have been purified to reduce or remove empty particles or particles having therapeutic agents, such as nucleic acids, associated with their outer surface.

[0344] The methods of the invention can be practiced in a variety of hosts. Preferred hosts include mammalian species such as primates (e.g., humans and chimpanzees as well as other non-human primates), dogs, cats, horses, cows, sheep, goats, rodents (e.g., rats and mice), rabbits, and pigs.

[0345] The amount of particles administered will depend on the ratio of therapeutic agent (e.g., nucleic acid) to lipid, the particular therapeutic agent (e.g., nucleic acid) used, the disease or disorder being treated, the age, weight, and condition of the patient, and the judgment of the clinician, but will generally be about 0.01 to about 50 mg / kg body weight, preferably about 0.1 to about 5 mg / kg body weight, or about 10 mg / kg per administration (e.g., injection). 8 ~10 10 It would be a particle.

[0346] In vitro administration For in vitro applications, delivery of therapeutic agents such as nucleic acids (e.g., interfering RNA or mRNA) can be to any cell grown in culture, whether of plant or animal origin, vertebrate or invertebrate, and of any tissue or type. In preferred embodiments, the cell is an animal cell, more preferably a mammalian cell, and most preferably a human cell.

[0347] When performed in vitro, contact between the cells and the lipid particles is performed in a biologically compatible medium. The concentration of the particles varies widely depending on the particular application, but is generally about 1 μmol to about 10 mmol. Treatment of the cells with the lipid particles is generally performed at physiological temperature (about 37° C.) for a period of about 1 to 48 hours, preferably about 2 to 4 hours.

[0348] In one group of preferred embodiments, the lipid particle suspension is dissolved in about 10 3 ~about 10 5 cells / ml, more preferably about 2×10 4 The suspension is added to cells plated at 60 to 80% confluence, with a cell density of 1000 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.

[0349] Endosomal Release Parameter (ERP) assays can be used to optimize the delivery efficiency of LNPs or other lipid particles of the present invention. ERP assays are described in detail in U.S. Patent Application Publication No. 20030077829, the disclosure of which is incorporated herein by reference in its entirety for all purposes. More specifically, the purpose of the ERP assay is to distinguish the effects of various cationic lipid and helper lipid components of LNPs based on their relative effects on endosomal membrane binding / engulfment or fusion with / destabilization of endosomal membranes. This assay allows for quantitative determination of how each component of LNPs or other lipid particles affects delivery efficiency, thereby optimizing LNPs or other lipid particles. Typically, ERP assays measure the expression of a reporter protein (e.g., luciferase, β-galactosidase, green fluorescent protein (GFP), etc.). In some cases, LNP formulations optimized for expression plasmids are also suitable for encapsulating interfering RNA or mRNA. In other cases, ERP assay can be adapted to measure the downregulation of the transcription or translation of target sequence in the presence or absence of interfering RNA (e.g., siRNA).In other cases, ERP assay can be adapted to measure the expression of target protein in the presence or absence of mRNA.By comparing the ERP for each of various LNPs or other lipid particles, it is easy to determine the optimized system, for example, the LNPs or other lipid particles that are most highly taken up into cells.

[0350] Cells for delivery of lipid particles The compositions and methods of the invention are used to treat a wide variety of cell types in vivo and in vitro. Suitable cells include, for example, hematopoietic progenitor (stem) cells, fibroblasts, keratinocytes, hepatocytes, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neural cells, resting lymphocytes, terminally differentiated cells, slow-cycling or 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., an interfering RNA (e.g., siRNA) or mRNA) is delivered to cancer cells, such as, for example, 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, appendix cancer cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, prostate cancer cells, renal cancer cells, cancer cells of the central nervous system, glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteogenic sarcoma tumor cells, and blood cancer cells.

[0351] 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 with cells of a wide variety of vertebrate animals, 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).

[0352] To the extent that it 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 references cited therein provide general guidance to cell culture. Cultured cell lines are often in the form of monolayers of cells, although cell suspensions are also used.

[0353] Detection of lipid particles In some embodiments, the lipid particles (e.g., LNPs) of the present invention are detectable in a subject at about 1, 2, 3, 4, 5, 6, 7, 8 hours or more. In other embodiments, the lipid particles (e.g., LNPs) of the present invention are detectable in a subject at about 8, 12, 24, 48, 60, 72, or 96 hours, or about 6, 8, 10, 12, 14, 16, 18, 19, 22, 24, 25, or 28 days after administration of the particles. The presence of the particles can be detected in cells, tissues, or other biological samples from a subject. The particles can be detected, for example, by direct detection of the particles, by detection of therapeutic nucleic acids such as interfering RNA (e.g., siRNA) or mRNA sequences, by detection of a target sequence of interest (i.e., by detecting expression or reduced expression of a sequence of interest), or by a combination thereof.

[0354] Particle detection The lipid particles of the present invention, such as LNPs, can be detected using any method known in the art. For example, labels can be directly or indirectly coupled to the components of the lipid particles using methods well known in the art. A wide variety of labels can be used, with the label being selected according to the required sensitivity, ease of conjugation with the lipid particle components, stability requirements, and available instrumentation and disposable provision. Suitable labels include spectroscopic labels such as fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon Green™; rhodamine and derivatives such as Texas Red, tetrarhodamine isothiocyanate (TRITC), digoxigenin, biotin, phycoerythrin, AMCA, CyDye™, etc.); 3 H, 125 I, 35 S, 14 C. 32 P, 33 Labels include, but are not limited to, radiolabels such as P; enzymes such as horseradish peroxidase, alkaline phosphatase; spectrocolorimetric labels such as colloidal gold or colored glass or plastic beads such as polystyrene, polypropylene, latex, etc. The labels can be detected using any means known in the art.

[0355] Nucleic Acid Detection Nucleic acids (e.g., interfering RNA or mRNA) are detected and quantified herein by any of several means well known to those skilled in the art. Detection of nucleic acids can be performed by well-known methods such as Southern analysis, Northern analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography. Additional analytical biochemical methods such as spectrophotometry, X-radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography can also be used.

[0356] The choice of nucleic acid hybridization format is not critical. A variety of nucleic acid hybridization formats are known to those skilled 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).

[0357] The sensitivity of hybridization assays can be improved through the use of nucleic acid amplification systems to increase the amount of target nucleic acid detected. In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known.Examples of techniques sufficient to guide one 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™), can be 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. Pat. No. 4,683,202, PCR Protocols, A Guide to Methods and Applications (Innis et al. eds.) Academic Press Inc. San Diego, Calif. (1990), Arnheim & Levinson (Oct. 1, 1990), C&EN 36; The Journal Of NIH Research,3:81(1991), Kwoh et al.,Proc.Natl.Acad.Sci.USA,86:1173(1989),Guatelli et al.,Proc.Natl.Acad.Sci.USA,87:1874(1990),Lomell et al. al., J.Clin.Chem.,35:1826(1989), Landegren et al.,Science,241:1077(1988), Van Brunt,Biotechnology,8:291(1990),Wu and Wallace,Gene,4:560(1989);Barringer et al. al., Gene, 89:117 (1990), and Sooknanan and Malek, Biotechnology, 13:563 (1995). An improved method for cloning in vitro amplified nucleic acids is described in US Pat. 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 if the PCR or LCR primers are designed to extend or ligate only if the selected sequence is present. Alternatively, selected sequences can be amplified, typically using, for example, non-specific PCR primers, and the amplified target region can then be probed for specific sequences that indicate mutations. The disclosures of the aforementioned references are incorporated herein by reference in their entirety for all purposes.

[0358] Nucleic acids, for example for use as probes in in vitro amplification methods, for use as gene probes, or as inhibitor moieties, are usually chemically synthesized according to the solid-phase phosphoramidite triester method described by Beaucage et al., Tetrahedron Letts., 22:1859 1862 (1981), for example using an automated synthesizer as described in Needham VanDevanter et al., Nucleic Acids Res., 12:6159 (1984). Purification of polynucleotides, if necessary, is usually performed either by native acrylamide gel electrophoresis, or by anion-exchange HPLC as described in Pearson et al., J. Chrom., 255:137 149 (1983). The sequence of synthetic polynucleotides 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.

[0359] An alternative method for measuring transcription levels 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 situ hybridization assays, cells are fixed to a solid support, usually a glass slide. When searching for DNA, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at moderate temperature to allow labeled specific probes to anneal. The probes are preferably labeled with radioisotopes or fluorescent reporters.

[0360] Working 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 easily recognize various non-critical parameters that can be changed or modified to produce essentially the same results. EXAMPLES

[0361] 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 easily recognize various non-critical parameters that can be changed or modified to produce essentially the same results.

[0362] Example 1: [ka] Synthesis of

[0363] (3-Chloropropyl)tris(decan-3-yloxy)silane (0.75 g, 1.21 mmol), dimethylamine (12.1 mmol (6.05 ml of 2.0 M in THF) and MeCN were heated in a sealed reaction vessel for 10 min at 150 °C in a microwave. After cooling, the reaction was partitioned between EtOAc and saturated NaHCO3. The organic layer was washed with water and brine, dried (Na2SO4) and concentrated under reduced pressure. The residue was purified by automated column chromatography (0-100% EtOAc / hexanes) to give N,N-dimethyl-3-(tris(decan-3-yloxy)silyl)propan-1-amine (0.16 g, 21.6%). 1H NMR CDCl3 δ 3.85 (m, 3H), 2.23 (m, 8H), 1.5 (m 15H), 1.28 (m, 34H), 0.87 (m, 19H), 0.55 (m, 2H).

[0364] The intermediate (3-chloropropyl)tris(decan-3-yloxy)silane was prepared as follows.

[0365] a. Preparation of (3-chloropropyl)tris(decan-3-yloxy)silane. [ka] Trichloro(3-chloropropyl)silane (1.72 g, 8.1 mmol) was stirred in Et2O at room temperature. A mixture of undecan-3-ol (4.19 g, 24.3 mmol) and TEA (3.15 g, 24.34 mmol) in Et2O was added dropwise at room temperature. The reaction was stirred at room temperature for 16 h. The resulting precipitate was removed by filtration and washed with additional Et2O. The organic layer was washed successively with NaHCO3, water, and brine, dried (Na2SO4), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (2% EtOAc / hexanes) to give (3-chloropropyl)tris(decan-3-yloxy)silane (2.48 g, 49.3%). 1H NMR CDCl3 δ 3.85 (p, 3H), 3.53 (t, 2H), 1.89 (m, 2H), 1.47 (m, 13H), 1.28 (m, 35H), 0.9 (m, 18H), 0.7 (m, 2H). [ka]

[0366] Step 2: Synthesis of N,N-dimethyl-3-(tris(decan-3-yloxy)silyl)propan-1-amine (4)

[0367] Examples 2 to 23 Using procedures similar to those described in Example 1, the following compounds (Examples 2-23) were prepared.

[0368] Example 2 [ka]

[0369] Example 3 [ka]

[0370] Example 4 [ka]

[0371] Example 5 [ka]

[0372] Example 6 [ka]

[0373] Example 7 [ka]

[0374] Example 8 [ka]

[0375] Example 9 [ka]

[0376] Example 10 [ka]

[0377] Example 11 [ka]

[0378] Example 12 [ka]

[0379] Example 13 [ka]

[0380] Example 14 [ka]

[0381] Example 15 [ka]

[0382] Example 16 [ka]

[0383] Example 17 [ka]

[0384] Example 18 [ka]

[0385] Example 19 [ka]

[0386] Example 20 [ka]

[0387] Example 21 [ka]

[0388] Example 22 [ka]

[0389] Example 23 [ka]

[0390] Example 24 Preparation of lipid formulations A lipid stock solution was prepared in 100% ethanol using the lipid identities and molar ratios described (total lipid content approximately 7 mg / mL). The mRNA was diluted with acetic acid at pH 5 and nuclease-free water to reach a concentration of 0.366 mg / mL mRNA in 100 mM acetic acid at pH 5. Equal volumes of each solution were mixed in a T-connector at 400 mL / min and diluted with approximately 4 volumes of PBS (pH 7.4) using the direct dilution method described in U.S. Pat. No. 9,404,127. The formulation was then placed in a Slide-A-Lyzer dialysis unit (MWCO 10,000) and dialyzed overnight against 10 mM Tris, 500 mM NaCl (pH 8) (Tris / NaCl buffer). After dialysis, the formulation was concentrated to approximately 0.6 mg / mL using a VivaSpin concentrator unit (MWCO 100,000) and then filtered through a 0.2 um syringe filter.

[0391] Example 24a The various lipids of the invention were mixed in the following molar ratios: DSPC (11%): cholesterol (33%): PEG 2000 -C-DMA (1.6%): Formulated into a LNP composition with Lipid X (55%). The composition comprises the following components: (a) Nucleic acid, (b) a mixture of cholesterol and DSPC; (c) Formula: [ka] where n is selected so that the resulting polymer chain has a molecular weight of about 2000 Daltons, and (d) Formula: [ka] Cationic lipids (wherein the conjugated lipid constituted about 1.5 mol % of the total lipid present in the particle, the DSPC constituted about 10 mol % of the total lipid present in the particle, the cholesterol constituted about 38.5 mol % of the total lipid present in the particle, and the cationic lipid constituted about 50 mol % of the total lipid present in the particle), Here, the lipid to nucleic acid ratio was about 19.6.

[0392] Example 24b A composition comprising the following ingredients: (a) Nucleic acid, (b) a mixture of cholesterol and DSPC; (c) Formula: [ka] where n is selected so that the resulting polymer chain has a molecular weight of about 2000 Daltons, and [ka] (d) Formula: Cationic lipids (wherein the conjugated lipid constituted about 1.6 mol % of the total lipid present in the particle, the DSPC constituted about 11 mol % of the total lipid present in the particle, the cholesterol constituted about 33 mol % of the total lipid present in the particle, and the cationic lipid constituted about 55 mol % of the total lipid present in the particle), Here, the lipid to nucleic acid ratio was approximately 20.5.

[0393] Example 24c A composition comprising the following ingredients: (a) Nucleic acid, (b) a mixture of cholesterol and DSPC; (c) Formula: [ka] where n is selected so that the resulting polymer chain has a molecular weight of about 2000 Daltons, and (d) Formula: [ka] Cationic lipids (wherein the conjugated lipid constituted about 1.6 mol % of the total lipid present in the particle, the DSPC constituted about 10.9 mol % of the total lipid present in the particle, the cholesterol constituted about 32.8 mol % of the total lipid present in the particle, and the cationic lipid constituted about 54.9 mol % of the total lipid present in the particle), Here, the lipid to nucleic acid ratio was approximately 20.2.

[0394] Example 25 In vivo assay Overall, LNPs were injected intravenously at 0.5 mg / kg into 5-8 week old female Balb / C mice and blood was collected 4-6 hours after dosing. Blood was collected in K2EDTA, processed to plasma and then stored frozen at -80 °C until analysis. Activity was assayed by testing plasma for expression of human EPO using either a human EPO ELISA kit from StemCell (catalog no. 01630) or R&D Systems (catalog DEP00) following the manufacturer's instructions. Data are shown in the table below.

[0395] As can be seen from the data in Table 1, several compositions are substantially more potent than the MC3 composition used in patisiran (Onpattro), an LNP product approved for the treatment of TTR amyloidosis. [Table 1-1] [Table 1-2]

[0396] Finally, preferred embodiments of the present invention are described in sections.

[0397] [Embodiment 1] 1. A nucleic acid-lipid particle comprising: (a) one or more nucleic acid molecules; (b) non-cationic lipids; (c) a conjugated lipid; and (d) a cationic lipid of formula (I): [ka] (In the formula, R 1 is C2~C 30 is a hydrocarbyl; R 2 is C2~C 30 is a hydrocarbyl; R 3 is C2~C 30 is a hydrocarbyl; X is a divalent C2-C8 alkyl; R 4 is NR a R b and; R a and R b are each independently selected from the group consisting of methyl, ethyl, propyl, cyclopropyl, and butyl, wherein methyl, ethyl, propyl, cyclopropyl, and butyl are optionally substituted with hydroxy; or R a and R b together with the nitrogen to which they are attached form an aziridine ring, an azetidine ring, a proline ring, a piperidine ring, a piperazine ring, or a morpholine ring, which ring is optionally substituted with hydroxyl or with C1-C6 alkyl optionally substituted with hydroxyl. wherein said one or more nucleic acid molecules are encapsulated within said lipid particle.

[0398] [Embodiment 2] R 1 But, C2~C 20 2. The nucleic acid-lipid particle of embodiment 1, which is hydrocarbyl.

[0399] [Embodiment 3] R 1 But, C2~C 15 2. The nucleic acid-lipid particle of embodiment 1, which is hydrocarbyl.

[0400] [Embodiment 4] R 1 But, C2~C 10 2. The nucleic acid-lipid particle of embodiment 1, which is hydrocarbyl.

[0401] [Embodiment 5] R 1 But C5~C 20 2. The nucleic acid-lipid particle of embodiment 1, which is hydrocarbyl.

[0402] [Embodiment 6] R 1 However, (C2~C 20 ) Alkyl, (C2-C 20 ) alkenyl, or (C2-C 20 2. The nucleic acid-lipid particle of embodiment 1, wherein R is 1 or 2.

[0403] [Embodiment 7] R 1 But C8~C 20 2. The nucleic acid-lipid particle of embodiment 1, wherein said nucleic acid-lipid particle is alkyl.

[0404] [Embodiment 8] R 1 But C8~C 20 2. The nucleic acid-lipid particle of embodiment 1, wherein said nucleic acid-lipid particle is alkenyl.

[0405] [Embodiment 9] R 1 But C8~C 20 2. The nucleic acid-lipid particle of embodiment 1, wherein said nucleic acid is alkynyl.

[0406] [Embodiment 10] R 1 C8-C 20 2. The nucleic acid-lipid particle of embodiment 1, wherein said nucleic acid-lipid particle is alkenyl.

[0407] [Embodiment 11] R 1 2. The nucleic acid-lipid particle of embodiment 1, wherein is (Z)-4-decen-1-yl, 1-nonyl, 3-undecyl, 1-decyl, 6(Z),15(Z)-henicosandiene-11-yl, 3-hexen-1-yl, 9(Z)-octadecen-1-yl, or 2-butyloct-1-yl, 4-(1-methylethenyl)cyclohexen-1-ylmethyl.

[0408] [Embodiment 12] R 2 But, C2~C 20 12. The nucleic acid-lipid particle of any one of embodiments 1 to 11, which is hydrocarbyl.

[0409] [Embodiment 13] R 2 But, C2~C 15 12. The nucleic acid-lipid particle of any one of embodiments 1 to 11, which is hydrocarbyl.

[0410] [Embodiment 14] R 2 But, C2~C 10 12. The nucleic acid-lipid particle of any one of embodiments 1 to 11, which is hydrocarbyl.

[0411] [Embodiment 15] R 2 But C5~C 20 12. The nucleic acid-lipid particle of any one of embodiments 1 to 11, which is hydrocarbyl.

[0412] [Embodiment 16] R 2 However, (C2~C 20 ) Alkyl, (C2-C 20 ) alkenyl, or (C2-C 20 12. The nucleic acid-lipid particle according to any one of embodiments 1 to 11, wherein R is 1 or 2.

[0413] [Embodiment 17] R2 However, (C8~C 20 12. The nucleic acid-lipid particle according to any one of embodiments 1 to 11, wherein the aryl group is aryl, aryl, or aryl.

[0414] [Embodiment 18] R 2 However, (C8~C 20 12. The nucleic acid-lipid particle according to any one of embodiments 1 to 11, wherein R is 1 or 2.

[0415] [Embodiment 19] R 2 However, (C8~C 20 12. The nucleic acid-lipid particle according to any one of embodiments 1 to 11, wherein R is 1 or 2.

[0416] [Embodiment 20] R 2 has only one double bond (C8-C 20 12. The nucleic acid-lipid particle according to any one of embodiments 1 to 11, wherein R is 1 or 2.

[0417] [Embodiment 21] R 2 The nucleic acid-lipid particle according to any one of embodiments 1 to 11, wherein is (Z)-4-decen-1-yl, 1-nonyl, 3-undecyl, 1-decyl, 6(Z),15(Z)-henicosanedien-11-yl, 3-hexen-1-yl, 9(Z)-octadecen-1-yl, or 2-butyloct-1-yl, 4-(1-methylethenyl)cyclohexen-1-ylmethyl.

[0418] [Embodiment 22] R 3 But, C2~C 20 22. The nucleic acid-lipid particle of any one of embodiments 1 to 21, which is hydrocarbyl.

[0419] [Embodiment 23] R 3 But, C2~C 15 22. The nucleic acid-lipid particle of any one of embodiments 1 to 21, which is hydrocarbyl.

[0420] [Embodiment 24] R 3 But, C2~C 10 22. The nucleic acid-lipid particle of any one of embodiments 1 to 21, which is hydrocarbyl.

[0421] [Embodiment 25] R 3 But C5~C 20 22. The nucleic acid-lipid particle of any one of embodiments 1 to 21, which is hydrocarbyl.

[0422] [Embodiment 26] R 3 However, (C2~C 20 ) Alkyl, (C2-C 20 ) alkenyl, or (C2-C 20 22. The nucleic acid-lipid particle according to any one of embodiments 1 to 21, wherein R is 1 or 2.

[0423] [Embodiment 27] R 3 However, (C8~C 20 22. The nucleic acid-lipid particle according to any one of embodiments 1 to 21, wherein the aryl group is aryl, aryl, or aryl.

[0424] [Embodiment 28] R 3 However, (C8~C 20 22. The nucleic acid-lipid particle according to any one of embodiments 1 to 21, wherein R is 1 or 2.

[0425] [Embodiment 29] R 3 However, (C8~C 20 22. The nucleic acid-lipid particle according to any one of embodiments 1 to 21, wherein R is 1 or 2.

[0426] [Embodiment 30] R 3 has only one double bond (C8-C 20 22. The nucleic acid-lipid particle according to any one of embodiments 1 to 21, wherein R is 1 or 2.

[0427] [Embodiment 31] R 322. The nucleic acid-lipid particle according to any one of embodiments 1 to 21, wherein is (Z)-4-decen-1-yl, 1-nonyl, 3-undecyl, 1-decyl, 6(Z),15(Z)-henicosanedien-11-yl, 3-hexen-1-yl, 9(Z)-octadecen-1-yl, adamantri-1-ylmethyl, 2-butyloct-1-yl, (Z)-2-((Z)-dec-4-en-1-yl)dodec-6-en-1-yl, or 4-(prop-1-en-2-yl)cyclohex-1-en-1-yl)methyl.

[0428] [Embodiment 32] 32. The nucleic acid-lipid particle of any one of embodiments 1 to 31, wherein X is a divalent C2 to C6 alkyl.

[0429] [Embodiment 33] 32. The nucleic acid-lipid particle of any one of embodiments 1 to 31, wherein X is a divalent C3 to C5 alkyl.

[0430] [Embodiment 34] 32. The nucleic acid-lipid particle of any one of embodiments 1 to 31, wherein X is -CH2CH2CH2-.

[0431] [Embodiment 35] 32. The nucleic acid-lipid particle of any one of embodiments 1 to 31, wherein X is -CH2CH2CH2CH2-.

[0432] [Embodiment 36] 32. The nucleic acid-lipid particle of any one of embodiments 1 to 31, wherein X is -CH2CH2CH2CH2CH2-.

[0433] [Embodiment 37] R a and R b is each independently selected from the group consisting of methyl, ethyl, propyl, cyclopropyl, and butyl, wherein methyl, ethyl, propyl, cyclopropyl, and butyl are optionally substituted with hydroxy.

[0434] [Embodiment 38] R a and R b together with the nitrogen to which they are attached form an aziridine ring, an azetidine ring, a proline ring, a piperidine ring, a piperazine ring, or a morpholine ring, which ring is optionally substituted with hydroxyl or with C1-C6 alkyl optionally substituted with hydroxyl.

[0435] [Embodiment 39] R a and R b are each independently selected from the group consisting of methyl and ethyl.

[0436] [Embodiment 40] R 4 The nucleic acid-lipid particle according to any one of embodiments 1 to 36, wherein is dimethylamino.

[0437] [Embodiment 41] the cationic lipid constitutes about 30 mol % to about 85 mol % of the total lipid present in the particle; the non-cationic lipid comprises from about 13 mol % to about 49.5 mol % of the total lipid present in the particle; The conjugated lipid comprises about 0.1 mol % to about 10 mol % of the total lipid present in the particle. 41. The nucleic acid-lipid particle according to any one of embodiments 1 to 40.

[0438] [Embodiment 42] the cationic lipid constitutes about 30 mol % to about 85 mol % of the total lipid present in the particle; the non-cationic lipid comprises from about 13 mol % to about 49.5 mol % of the total lipid present in the particle; 41. The nucleic acid-lipid particle of any one of embodiments 1 to 40, wherein the conjugated lipid comprises from about 0.1 mol % to about 10 mol % of the total lipid present in the particle.

[0439] [Embodiment 43] 43. The nucleic acid-lipid particle according to any one of embodiments 1 to 42, wherein the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, viral RNA (vRNA), self-amplifying RNA, and combinations thereof.

[0440] [Embodiment 44] 44. The nucleic acid-lipid particle of embodiment 43, wherein the nucleic acid is an mRNA molecule.

[0441] [Embodiment 45] 44. The nucleic acid-lipid particle of embodiment 43, wherein the nucleic acid comprises a double-stranded siRNA molecule.

[0442] [Embodiment 46] 46. ​​The nucleic acid-lipid particle of embodiment 45, wherein the double-stranded siRNA molecule comprises at least one modified nucleotide.

[0443] [Embodiment 47] 47. The nucleic acid-lipid particle of embodiment 46, wherein the siRNA comprises at least one 2'-O-methyl (2'OMe) nucleotide.

[0444] [Embodiment 48] 48. The nucleic acid-lipid particle of any one of embodiments 1 to 47, wherein the non-cationic lipid comprises cholesterol or a derivative thereof.

[0445] [Embodiment 49] 49. The nucleic acid-lipid particle of embodiment 48, wherein said cholesterol or derivative thereof constitutes from about 31.5 mol % to about 42.5 mol % of the total lipid present in said particle.

[0446] [Embodiment 50] 48. The nucleic acid-lipid particle of any one of embodiments 1 to 47, wherein the non-cationic lipid comprises a phospholipid.

[0447] [Embodiment 51] 48. The nucleic acid-lipid particle of any one of embodiments 1 to 47, wherein the non-cationic lipid comprises a mixture of phospholipids and cholesterol or a derivative thereof.

[0448] [Embodiment 52] 52. The nucleic acid-lipid particle of embodiment 51, wherein the phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or a mixture thereof.

[0449] [Embodiment 53] the phospholipid constitutes about 4 mol % to about 10 mol % of the total lipid present in the particle; The cholesterol constitutes about 30 mol % to about 40 mol % of the total lipids present in the particles. 52. The nucleic acid-lipid particle of embodiment 51.

[0450] [Embodiment 54] the phospholipid constitutes about 10 mol % to about 30 mol % of the total lipid present in the particle; The cholesterol constitutes about 10 mol % to about 30 mol % of the total lipid present in the particle. 52. The nucleic acid-lipid particle of embodiment 51.

[0451] [Embodiment 55] 55. The nucleic acid-lipid particle of any one of embodiments 1 to 54, wherein the conjugated lipid comprises a polyethylene glycol (PEG)-lipid conjugate.

[0452] [Embodiment 56] 56. The nucleic acid-lipid particle of embodiment 55, wherein the PEG-lipid conjugate comprises a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, or a mixture thereof.

[0453] [Embodiment 57] 57. The nucleic acid-lipid particle of embodiment 56, wherein the PEG-DAA conjugate comprises a PEG-dimyristyloxypropyl (PEG-DMA) conjugate, a PEG-distearyloxypropyl (PEG-DSA) conjugate, or a mixture thereof.

[0454] [Embodiment 58] 58. The nucleic acid-lipid particle of embodiment 57, wherein the PEG-DAA conjugate comprises a PEG-dimyristyloxypropyl (PEG-DMA) conjugate.

[0455] [Embodiment 59] The polyethylene glycol (PEG)-lipid conjugate has the formula: [ka] [In the formula, A is (C1-C6)alkyl, (C3-C8)cycloalkyl, (C3-C8)cycloalkyl(C1-C6)alkyl, (C1-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (C1-C6)alkanoyl, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, or (C2-C6)alkanoyloxy; wherein any of the (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; B is a polyethylene glycol chain having a molecular weight of about 550 daltons to about 10,000 daltons; C is -LR a (L is a direct bond, -C(O)O-, -C(O)NR b -, -NR b -, -C(O)-, -NR b C(O)O-, -NR b C(O)NR b selected from the group consisting of -, -SS-, -O-, -(O)CCH2CH2C(O)-, and -NHC(O)CH2CH2C(O)NH-; R a is a branched (C 10 ~C 50 ) alkyl or branched (C 10 ~C 50 ) alkenyl, wherein the branched (C 10 ~C 50 ) alkyl or branched (C 10 ~C 50 ) one or more carbon atoms of the alkenyl is replaced with -O-; R b are each independently H or (C1-C6) alkyl). or a salt thereof.

[0456] [Embodiment 60] 60. The nucleic acid-lipid particle of any one of embodiments 55 to 59, wherein the PEG has an average molecular weight of about 2,000 daltons.

[0457] [Embodiment 61] 61. The nucleic acid-lipid particle of any one of embodiments 1 to 60, wherein the conjugated lipid comprises from about 1 mol % to about 2 mol % of the total lipid present in the particle.

[0458] [Embodiment 62] 62. The nucleic acid-lipid particle of any one of embodiments 1 to 61, wherein the nucleic acid in the nucleic acid-lipid particle is not substantially degraded after incubating the particle in serum at 37° C. for 30 minutes.

[0459] [Embodiment 63] 63. The nucleic acid-lipid particle of any of the preceding embodiments, wherein the nucleic acid is fully encapsulated within the nucleic acid-lipid particle.

[0460] [Embodiment 64] 64. The nucleic acid-lipid particle of any of the preceding embodiments, wherein the nucleic acid-lipid particle has a lipid:nucleic acid mass ratio of about 5 to about 15.

[0461] [Embodiment 65] 64. The nucleic acid-lipid particle of any of the preceding embodiments, wherein the nucleic acid-lipid particle has a lipid:nucleic acid mass ratio of about 5 to about 30.

[0462] [Embodiment 66] 66. The nucleic acid-lipid particle according to any one of embodiments 1 to 65, wherein the nucleic acid-lipid particle has a median diameter of about 40 nm to about 150 nm.

[0463] [Embodiment 67] the cationic lipid constitutes about 56.5 mol % to about 66.5 mol % of the total lipid present in the particle; The non-cationic lipid comprises cholesterol or a derivative thereof, which constitutes about 31.5 mol % to about 42.5 mol % of the total lipid present in the particle; The PEG-lipid conjugate constitutes about 1 mol% to about 2 mol% of the total lipid present in the particle; 41. The nucleic acid-lipid particle according to any one of embodiments 1 to 40.

[0464] [Embodiment 68] 68. The nucleic acid-lipid particle of embodiment 67, wherein the nucleic acid-lipid particle comprises about 61.5 mol % cationic lipid, about 36.9 mol % cholesterol or a derivative thereof, and about 1.5 mol % PEG-lipid conjugate.

[0465] [Embodiment 69] the cationic lipid constitutes about 52 mol % to about 62 mol % of the total lipid present in the particle; the non-cationic lipid comprises a mixture of phospholipids and cholesterol or a derivative thereof, the mixture constituting about 36 mol % to about 47 mol % of the total lipid present in the particle; The PEG-lipid conjugate constitutes about 1 mol% to about 2 mol% of the total lipid present in the particle; 41. The nucleic acid-lipid particle according to any one of embodiments 1 to 40.

[0466] [Embodiment 70] 70. The nucleic acid-lipid particle of embodiment 69, wherein the nucleic acid-lipid particle comprises about 57.1 mol % cationic lipid, about 7.1 mol % phospholipid, about 34.3 mol % cholesterol or a derivative thereof, and about 1.4 mol % PEG-lipid conjugate.

[0467] [Embodiment 71] 70. The nucleic acid-lipid particle of embodiment 69, wherein the nucleic acid-lipid particle comprises about 57.1 mol % cationic lipid, about 20 mol % phospholipid, about 20 mol % cholesterol or a derivative thereof, and about 1.4 mol % PEG-lipid conjugate.

[0468] [Embodiment 72] The cationic lipid constitutes 50 mol % to 65 mol % of the total lipid present in the particle, The non-cationic lipid comprises a mixture of a phospholipid and cholesterol or a derivative thereof (wherein the phospholipid constitutes 4 mol% to 10 mol% of the total lipid present in the particle, and the cholesterol or a derivative thereof constitutes 30 mol% to 40 mol% of the total lipid present in the particle), The conjugated lipid comprises 0.5 mol% to 2 mol% of the total lipid present in the particle; 41. The nucleic acid-lipid particle according to any one of embodiments 1 to 40.

[0469] [Embodiment 73] The cationic lipid constitutes 50 mol % to 65 mol % of the total lipid present in the particle, The non-cationic lipid comprises a mixture of a phospholipid and cholesterol or a derivative thereof (wherein the phospholipid constitutes 3 mol% to 15 mol% of the total lipid present in the particle, and the cholesterol or a derivative thereof constitutes 30 mol% to 40 mol% of the total lipid present in the particle), The conjugated lipid comprises 0.5 mol% to 2 mol% of the total lipid present in the particle; 41. The nucleic acid-lipid particle according to any one of embodiments 1 to 40.

[0470] [Embodiment 74] The cationic lipid constitutes 50 mol % to 65 mol % of the total lipid present in the particle, the non-cationic lipid comprises up to 49.5 mol% of the total lipid present in the particle; Cholesterol or a derivative thereof constitutes 30 mol % to 40 mol % of the total lipid present in the particle; The conjugated lipid comprises 0.5 mol% to 2 mol% of the total lipid present in the particle; 41. The nucleic acid-lipid particle according to any one of embodiments 1 to 40.

[0471] [Embodiment 75] The cationic lipid constitutes 50 mol % to 85 mol % of the total lipid present in the particle, the non-cationic lipid constitutes 13 mol % to 49.5 mol % of the total lipid present in the particle; The conjugated lipid comprises 0.5 mol% to 2 mol% of the total lipid present in the particle; 41. The nucleic acid-lipid particle according to any one of embodiments 1 to 40.

[0472] [Embodiment 76] The cationic lipid or a salt thereof constitutes about 30 mol % to about 50 mol % of the total lipid present in the particle, the non-cationic lipid comprises a mixture of phospholipids and cholesterol or a derivative thereof, the mixture constituting about 47 mol % to about 69 mol % of the total lipid present in the particle; The conjugated lipid comprises about 1 mol % to about 3 mol % of the total lipid present in the particle. 41. The nucleic acid-lipid particle according to any one of embodiments 1 to 40.

[0473] [Embodiment 77] The non-cationic lipid comprises a mixture of cholesterol and DSPC, and the conjugated lipid comprises [ka] where n is selected so that the resulting polymer chain has a molecular weight of about 2000 Daltons, and said cationic lipid is [ka] and and wherein the conjugated lipid constitutes about 2 mol % of the total lipid present in the particle; the DSPC constitutes about 10 mol % of the total lipid present in the particle; the cholesterol constitutes about 48 mol% of the total lipid present in the particle; The cationic lipid comprises about 40 mol% of the total lipid present in the particle. 2. The nucleic acid-lipid particle of embodiment 1.

[0474] [Embodiment 78] The non-cationic lipid comprises a mixture of cholesterol and DSPC, and the conjugated lipid comprises [ka] where n is selected so that the resulting polymer chain has a molecular weight of about 2000 Daltons, and said cationic lipid is [ka] and and wherein the conjugated lipid constitutes about 2 mol % of the total lipid present in the particle; the DSPC constitutes about 10 mol % of the total lipid present in the particle; the cholesterol constitutes about 48 mol% of the total lipid present in the particle; The cationic lipid comprises about 40 mol% of the total lipid present in the particle. 2. The nucleic acid-lipid particle of embodiment 1.

[0475] [Embodiment 79] The non-cationic lipid comprises a mixture of cholesterol and DSPC, and the conjugated lipid comprises [ka] where n is selected so that the resulting polymer chain has a molecular weight of about 2000, and said cationic lipid is [ka] and wherein the conjugated lipid comprises about 1.6 mol % of the total lipid present in the particle; the DSPC constitutes about 10.9 mol % of the total lipid present in the particle; the cholesterol constitutes about 32.8 mol% of the total lipid present in the particle; The cationic lipid comprises about 54.9 mol% of the total lipid present in the particle. 2. The nucleic acid-lipid particle of embodiment 1.

[0476] [Embodiment 80] 80. The nucleic acid-lipid particle of any one of embodiments 77 to 79, wherein the lipid to nucleic acid ratio is about 24.

[0477] [Embodiment 81] A pharmaceutical composition comprising the nucleic acid-lipid particle according to any one of embodiments 1 to 80 and a pharma- ceutically acceptable carrier.

[0478] [Embodiment 82] 1. A method for introducing a nucleic acid into a cell, comprising: The method comprising contacting the cell with a nucleic acid-lipid particle according to any of embodiments 1 to 80 or a composition according to embodiment 81.

[0479] [Embodiment 83] 83. The method of embodiment 82, wherein the cell is in a mammal.

[0480] [Embodiment 84] 1. A method for in vivo delivery of a nucleic acid, comprising: The method comprises administering to a mammalian subject a nucleic acid-lipid particle according to any of embodiments 1 to 80 or a composition according to embodiment 81.

[0481] [Embodiment 85] 85. The method of embodiment 84, wherein said administration is selected from the group consisting of oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, and intradermal.

[0482] [Embodiment 86] A nucleic acid-lipid particle according to any of embodiments 1 to 80 or a composition according to embodiment 81 for use in vivo delivery of a nucleic acid to a mammal.

[0483] [Embodiment 87] Use of a nucleic acid-lipid particle according to any of embodiments 1 to 80 or a composition according to embodiment 81 for the preparation of a medicament for the in vivo delivery of a nucleic acid to a mammal.

[0484] [Embodiment 88] 1. A method for treating a disease or disorder in a mammalian subject in need of such treatment, comprising: The method comprising administering to the mammalian subject a therapeutically effective amount of a nucleic acid-lipid particle according to any one of embodiments 1 to 80 or a composition according to embodiment 81.

[0485] [Embodiment 89] 89. The method of embodiment 88, wherein the disease or disorder is selected from the group consisting of viral infection, liver disease or disorder, and cancer.

[0486] [Embodiment 90] A nucleic acid-lipid particle according to any of embodiments 1 to 80 or a composition according to embodiment 81 for use in treating a disease or disorder in a mammal.

[0487] [Embodiment 91] Use of a nucleic acid-lipid particle according to any of embodiments 1 to 80 or a composition according to embodiment 81 for the preparation of a medicament for the treatment of a disease or disorder in a mammal.

[0488] [Embodiment 92] 41. A compound of formula (I) or a salt thereof according to any one of embodiments 1 to 40.

[0489] [Embodiment 93] A composition comprising a compound of formula (I) as defined in any one of embodiments 1 to 40, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0490] [Embodiment 94] 94. The composition of embodiment 93, further comprising a nucleic acid.

[0491] [Embodiment 95] 41. A lipid nanoparticle comprising a cationic lipid of formula (I) according to any one of embodiments 1 to 40.

[0492] [Embodiment 96] Compounds of formula (I) according to any one of embodiments 1 to 40: [ka] A process for preparing a corresponding compound of formula (Ia): [ka] (In the formula, R 4a is bromo or chloro) to the formula HNR a R b to obtain a compound of formula (I), 4 is NR a R b wherein

[0493] [Embodiment 97] The compound of formula (Ia) 1 , R 2 , and R 3 are the same C2~C 30 hydrocarbyl) to the corresponding compound of formula (Ib): [ka] (wherein each Z is independently bromo or chloro), and C2-C 30 97. The method of embodiment 96, further comprising preparing the compound of formula (Ia) by reacting with a hydrocarbyl-OH alcohol.

[0494] [Embodiment 98] Compounds of formula (I): [ka] (In the formula, R 1 is C2~C 30 is a hydrocarbyl; R 2 is C2~C 30 is a hydrocarbyl; R 3 is C2~C 30 is a hydrocarbyl; X is a divalent C2-C8 alkyl; R 4 is NR a R b and; R a and R b are each independently selected from the group consisting of methyl, ethyl, propyl, cyclopropyl, and butyl, wherein methyl, ethyl, propyl, cyclopropyl, and butyl are optionally substituted with hydroxy; or R a and R b together with the nitrogen to which they are attached form an aziridine ring, azetidine ring, proline ring, piperidine ring, piperazine ring, or morpholine ring, which is optionally substituted with hydroxyl or C1-C6 alkyl optionally substituted with hydroxyl.

[0495] [Embodiment 99] A composition comprising a compound of formula (I) as defined in embodiment 98 or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable carrier.

Claims

[Claim 1] 1. A nucleic acid-lipid particle comprising: (a) one or more nucleic acid molecules; (b) a non-cationic lipid; (c) a conjugated lipid; and (d) A cationic lipid of formula (I): 【Chemistry 1】 (In the formula, R １ is C ２ ~C ３０ is hydrocarbyl; R ２ is C ２ ~C ３０ is hydrocarbyl; R ３ is C ２ ~C ３０ is hydrocarbyl; X is a divalent C ２ ~C ８ is alkyl; R ４ is N.R. ａ R ｂ and R ａ and R ｂ are each independently selected from the group consisting of methyl, ethyl, propyl, cyclopropyl, and butyl, wherein methyl, ethyl, propyl, cyclopropyl, and butyl are optionally substituted with hydroxy; or R ａ and R ｂ together with the nitrogen to which they are attached form an aziridine ring, an azetidine ring, a proline ring, a piperidine ring, a piperazine ring, or a morpholine ring, which ring is a C substituted with hydroxyl or optionally with hydroxy. １ ~C ６ alkyl, optionally substituted wherein said one or more nucleic acid molecules are encapsulated within said lipid particle.

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