Ionizable cationic lipids for lipid nanoparticles

Abstract

Certain embodiments of the present invention provide ionizable lipids with optimized clearance properties. Certain embodiments of the present invention also provide nucleic acid-lipid particles comprising ionizable lipids, methods of making the lipid particles, and methods of delivering and / or administering the lipid particles.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Priority This application claims priority to U.S. Provisional Patent Application No. 63 / 305,181, filed January 31, 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Ionizable cationic lipids are typically the primary lipid component of lipid nanoparticles (LNPs). They are designed to be charge-neutral at standard physiological pH (approximately 7) but acquire a positive charge at lower (acidic) pH. This aids in the encapsulation and intracellular delivery of nucleic acid payloads during LNP formation, as the endosomal fusion process requires the positive charge of ionizable lipids.

[0003] Ionizable lipids such as MC3 (used in the FDA-approved siRNA-LNP product Onpattro) and 3D-P-DMA have demonstrated good activity and tolerability in a variety of applications. However, they are not rapidly metabolized and take several weeks to be cleared from target tissues. This makes them less suitable for applications requiring multiple administrations, especially those with short dosing intervals and / or chronic indications.

[0004] Currently, there is a need for ionizable lipids that exhibit improved clearance while retaining good activity in their ability to deliver nucleic acids. There is also a need for ionizable lipids that have beneficial or improved biodegradability. Summary of the Invention [Means for solving the problem]

[0005] The present invention provides ionizable lipids with improved biodegradability and / or clearance rates.

[0006] In one embodiment, the present invention provides a compound of formula (I): [ka] or a salt thereof, wherein: X is -CH2-, -O-, -O(C=O); or -C(=O)-; Y is -CH2-, -O-, -SO2-, or -C(=O)-; R 1 is C2-C 25 is a hydrocarbyl; R 2 is C1-C3 alkyl, and R 3 is C1-C3 alkyl, or R 2 and R 3 together with the nitrogen to which they are attached form an aziridino, azetidino, or pyrrolidino ring; m is 0, 1, 2, 3, 4, 5, or 6; n is 0 to 15; o is 0 to 15; L 1 does not exist, -(C=O)-O-, -(C=O)N(H)N(R 4 )-, -(C=O)N(H)-N=C(R 4 )-, -(C=O)N(H)N(H)C(R 4 )(R 5 )-, -(C=O)-O-CH2-R z , or -O(C=O)-R w - and; L 2 does not exist, -(C=O)-O-, -(C=O)N(H)N(R 4’ )-, -(C=O)N(H)-N=C(R 4’ )-, -(C=O)N(H)N(H)C(R 4’ )(R 5’ )-, -(C=O)-O-CH2-R z’ , or -O(C=O)-R x - and; a is H or F; b is H or F; c is H or F; d is H or F; or c and d together with the carbon to which they are attached form -C(=O)-; R 4 is H or C2-C 25 is a hydrocarbyl; R 5 is H or C2-C 25 is a hydrocarbyl; R 4’ is H or C2-C 25 is a hydrocarbyl; R 5’ is H or C2-C 25 is a hydrocarbyl; R 6 is C2-C 25 is a hydrocarbyl; R 7 is C2-C 25 is a hydrocarbyl; R w is absent, -C(F)H-, or -C(F)2-; R x is absent, -C(F)H-, or -C(F)2-; R z is C2-C 10 phenyl substituted with 1, 2, or 3 groups independently selected from hydrocarbyl; R z’ is C2-C 10 and phenyl substituted with 1, 2, or 3 groups independently selected from hydrocarbyl.

[0007] The present invention also provides compositions comprising 1) an ionizable lipid of the present invention and 2) an active or therapeutic agent.

[0008] The present invention also provides lipid nanoparticles comprising the ionizable lipids of the present invention.

[0009] The present invention also provides lipid nanoparticles comprising (a) one or more active or therapeutic agents; (b) an ionizable lipid of the present invention; (c) a non-cationic lipid; and (d) a conjugated lipid. Typically, the agent or agents are encapsulated within the lipid nanoparticle.

[0010] The present invention also provides pharmaceutical compositions comprising 1) an ionizable lipid of the present invention and 2) one or more active or therapeutic agents.

[0011] The present invention also provides a pharmaceutical composition comprising the lipid nanoparticles of the present invention and a pharmaceutically acceptable carrier.

[0012] The present invention also provides a method of delivering an active or therapeutic agent (eg, a nucleic acid) to an animal, comprising administering to the animal a nanoparticle of the present invention.

[0013] The present invention also provides methods for introducing nucleic acids into cells, comprising contacting the cells in vivo, ex vivo, or in vitro with the nucleic acid-lipid particles described herein.

[0014] The present invention also provides a method of 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.

[0015] The invention also provides processes and intermediates disclosed herein that are useful for making the compounds, formulations, or nanoparticles of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0016] definition As used herein, the following terms have the meanings set forth below unless specified otherwise.

[0017] As used herein, the term "hydrocarbyl" describes an organic compound or radical consisting of about 4 to about 500 atoms selected from carbon and hydrogen. Certain specific hydrocarbyl moieties have a specified number of carbon atoms, e.g., C2-C 25Hydrocarbyls are hydrocarbons having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon atoms and up to 52 hydrogen atoms, depending on the degree of unsaturation. Hydrocarbyl moieties include, but are not limited to, branched or unbranched alkyl, branched or unbranched alkenyl, branched or unbranched alkynyl, cycloalkyl, and aryl moieties. In one embodiment, the hydrocarbyl comprises from about 4 to about 400 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl comprises from about 4 to about 300 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl comprises from about 4 to about 200 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl comprises from about 4 to about 100 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl comprises from about 4 to about 62 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl comprises from about 20 to about 500 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl comprises from about 20 to about 400 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl comprises from about 20 to about 300 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl comprises from about 20 to about 200 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl comprises from about 20 to about 100 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl comprises from about 20 to about 62 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl comprises from about 40 to about 500 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl comprises from about 40 to about 400 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl comprises from about 40 to about 300 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl comprises from about 40 to about 200 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl consists of from about 40 to about 100 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl consists of from about 40 to about 62 atoms selected from carbon and hydrogen.In one embodiment, the hydrocarbyl consists of at least about 20, at least about 40, at least about 60, or at least about 80 atoms selected from carbon and hydrogen. In one embodiment, the hydrocarbyl consists of fewer than about 500, fewer than about 400, fewer than about 300, fewer than about 200, or fewer than about 100 atoms selected from carbon and hydrogen.

[0018] The term "alkyl," by itself or as part of another substituent, unless otherwise stated, means an alkyl group having the indicated number of carbon atoms (i.e., C 1-8 means 1 to 8 carbons) straight or branched chain hydrocarbon radicals. Examples include (C1-C8) alkyl, (C2-C8) alkyl, C1-C6) alkyl, (C2-C6) alkyl, and (C3-C6) alkyl. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and higher homologs and isomers.

[0019] The term "alkenyl" refers to an unsaturated alkyl radical having one or more (e.g., 1, 2, 3, or 4) double bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), and higher homologs and isomers.

[0020] The term "alkynyl" refers to an unsaturated alkyl radical having one or more (e.g., 1, 2, 3, or 4) triple bonds. Examples of such unsaturated alkyl groups include ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers.

[0021] The term "alkoxy" refers to an alkyl group linked to the remainder of the molecule via an oxygen atom ("oxy").

[0022] The term "alkylthio" refers to an alkyl group linked to the remainder of the molecule via a thio group.

[0023] The term "cycloalkyl" refers to any saturated or partially unsaturated (non-aromatic) carbocyclic ring (i.e., (C3-C8)carbocycle) having 3 to 8 carbon atoms. The term also includes saturated, all-carbon, fused ring systems (e.g., ring systems containing two, three, or four carbocycles). Thus, carbocycle includes bicyclic carbocycles (e.g., bicyclic carbocycles having about 3 to 15 carbon atoms, about 6 to 15 carbon atoms, or 6 to 12 carbon atoms, such as bicyclo[3.1.0]hexane and bicyclo[2.1.1]hexane) and polycyclic carbocycles (e.g., tricyclic and tetracyclic carbocycles having up to about 20 carbon atoms). Rings in fused ring systems can be connected to each other through fused, spiro, and bridged bonds, where valency requirements permit. For example, polycyclic carbocycles can be linked together through a single carbon atom to form a spiro bond (e.g., spiropentane, spiro[4,5]decane, etc.), through two adjacent carbon atoms to form a fused bond (e.g., carbocycles such as decahydronaphthalene, norsabinane, and norcarane), or through two nonadjacent carbon atoms to form a bridged bond (e.g., norbornane, bicyclo[2.2.2]octane, etc.). Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptane, pinane, and adamantane.

[0024] The term "aryl," as used herein, refers to a single all-carbon aromatic ring or a multiple fused all-carbon ring system in which at least one of the rings is aromatic. For example, in certain embodiments, aryl groups have 6 to 20 carbon atoms, 6 to 14 carbon atoms, 6 to 12 carbon atoms, or 6 to 10 carbon atoms. Aryl includes the phenyl radical. Aryl also includes multiple fused carbocyclic ring systems (e.g., ring systems containing 2, 3, or 4 rings) having about 9 to 20 carbon atoms in which at least one ring is aromatic, and the other rings may or may not be aromatic (i.e., cycloalkyl). The rings of such multiple fused ring systems may be connected to each other via fused, spiro, and bridged bonds, as permitted by valency requirements. As defined above, it should be understood that the point of attachment of multiple fused ring systems can be at any position on the ring system, including the aromatic or carbocyclic portions of the rings. Non-limiting examples of aryl groups include, but are not limited to, phenyl, indenyl, indanyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, and the like.

[0025] 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., duplex RNA such as siRNA, aiRNA, or pre-miRNA) that can reduce or inhibit expression of a targeted gene or sequence (e.g., by mediating degradation of or inhibiting translation of mRNA complementary to the interfering RNA sequence) when the interfering RNA is present in the same cell as the targeted 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 the targeted gene or sequence, or may contain regions of mismatch (i.e., mismatch motifs). The sequence of the interfering RNA can correspond to the full-length target gene or a subsequence thereof.

[0026] Interfering RNAs include "small interfering RNAs" or "siRNAs," e.g., interfering RNAs that are about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more usually about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, and preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., where each complementary sequence in the double-stranded siRNA is about 15-60, 15-50, or 15-40 (duplex) nucleotides in length). The siRNA duplex may be about 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 siRNA duplex may be 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 may 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 constructed from two separate molecules, one strand being the sense strand and the other being the complementary antisense strand; double-stranded polynucleotide molecules constructed from single-stranded molecules, in which the sense and antisense regions are connected by a nucleic acid-based or non-nucleic acid-based linker; double-stranded polynucleotide molecules with a hairpin secondary structure having 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, wherein the circular polynucleotide can be processed in vivo or in vitro to generate active double-stranded siRNA molecules.

[0027] Preferably, siRNAs are chemically synthesized. siRNAs can also be generated by cleavage of long dsRNAs (e.g., dsRNAs greater than about 25 nucleotides in length) with E. coli RNase III or Dicer. These enzymes process dsRNA into biologically active siRNA (see, for example, Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al., Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); and Robertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, dsRNA is at least 50 nucleotides to about 100, 200, 300, 400 or 500 nucleotides in length.DsRNA can be 1000, 1500, 2000, 5000 nucleotides or more in length.DsRNA can encode the whole or partial gene transcript.In certain cases, siRNA can be encoded by plasmid (for example, transcribed as the sequence that spontaneously folds into a double strand with hairpin loop).

[0028] 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 is not 100% complementary to the target sequence. An interfering RNA may have at least one, two, three, four, five, six, or more mismatch regions. The mismatch regions may be adjacent or separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. A mismatch motif or mismatch region may comprise a single nucleotide or may comprise 2, 3, 4, 5, or more nucleotides.

[0029] An "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent, such as a nucleic acid (e.g., interfering RNA or mRNA), is an amount sufficient to produce a desired effect, for example, an amount sufficient to cause inhibition of the expression of a target sequence compared to the normal expression level detected in the absence of interfering RNA, or an amount sufficient to cause mRNA-induced expression of a certain amount of protein that causes a desired biological effect in the organism in which the protein is expressed. Inhibition of the expression of a target gene or target sequence is achieved when the value obtained using interfering RNA is about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% compared to 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.

[0030] "Reducing," "reducing," "reducing," or "reducing" an immune response induced 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 reduction in an immune response induced by a modified interfering RNA may be determined relative to the level of the immune response in the presence of unmodified interfering RNA. A detectable reduction can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more lower than the immune response detected in the presence of unmodified interfering RNA. A reduction in the immune response to an interfering RNA is typically measured by a decrease in cytokine production (e.g., IFNγ, IFNα, TNFα, IL-6, or IL-12) by responder cells in vitro or by a decrease in cytokine production in the serum of a mammalian subject after administration of the interfering RNA.

[0031] "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 reduction in the immune response to a modified mRNA can be determined relative to the level of the immune response in the presence of 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 unmodified mRNA. A reduction in the immune response to an mRNA is typically measured by a decrease in cytokine production (e.g., IFNγ, IFNα, TNFα, IL-6, or IL-12) by responder cells in vitro or in the serum of a mammalian subject following administration of the mRNA.

[0032] As used herein, the term "responder cell" refers to a cell, preferably a mammalian cell, that generates 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, for example, the production of cytokines or growth factors, such as TNF-α, IFN-α, IFN-β, IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TGF, and combinations thereof.

[0033] "Substantial identity" refers to a sequence that hybridizes to a reference sequence under stringent conditions or has a specified percent identity over a specified region of the reference sequence.

[0034] The phrase "stringent hybridization conditions" refers to conditions under which a nucleic acid will hybridize to its target sequence but to no other sequences, usually in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will vary under different circumstances. Longer sequences hybridize more readily, 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). Generally, stringent conditions are determined by the thermal melting point (T) of a particular sequence at a defined ionic strength pH. m ) is selected to be approximately 5 to 10°C lower than T mis the temperature (under defined ionic strength, pH, and nuclear concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (when the target sequence is present in excess, T m (At equilibrium, 50% of the probes are occupied.) Stringent conditions may 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.

[0035] 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, followed by a wash in 0.2xSSC and 0.1% SDS at 65°C. For PCR, a temperature of about 36°C is typical for low-stringency amplification, although annealing temperatures can vary from about 32°C to 48°C depending on primer length. For high-stringency PCR amplification, a temperature of about 62°C is typical, although 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 described, for example, in Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. NY (1990).

[0036] 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 typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include hybridization at 37°C in a buffer of 40% formamide, 1M NaCl, and 1% SDS, and washing in 1x SSC at 45°C. Positive hybridization is at least twice the background. Those skilled in the art will readily recognize that alternative hybridization and washing conditions can be used to achieve 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.

[0037] 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 specified percentage of the same nucleotides (i.e., at least about 60%, preferably at least about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region) when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of a sequence, 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.

[0038] In sequence comparison, one sequence usually serves as a reference sequence, and test sequences are compared to it.When using sequence comparison algorithm, test sequences and reference sequences are input into computer, and subsequence coordinates are designated as needed, and parameters of sequence algorithm program 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 sequences to reference sequences based on program parameters.

[0039] As used herein, a "comparison window" includes reference to any one section of contiguous positions selected from the group consisting of about 5 to about 60, usually about 10 to about 45, and more usually about 15 to about 30, and may be used to compare a sequence to a reference sequence of the same number of contiguous positions after optimally aligning 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 similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds. (1995 supplement)).

[0040] Preferred examples of algorithms suitable for determining percent sequence identity and sequence similarity are 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 acid of the present invention, BLAST and BLAST 2.0 are used with the parameters described herein.Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0041] The 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 of the similarity measures provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences occurs by chance. For example, if the smallest sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered to be similar to the reference sequence.

[0042] 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, including 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 nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and have similar binding properties to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (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 substituted 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 to one another through phosphate groups. "Base" includes purines and pyrimidines, which further include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogs, 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.

[0043] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises partial or full-length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

[0044] As used herein, "gene product" refers to the product of a gene, such as an RNA transcript or a polypeptide.

[0045] The term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, characterized by being insoluble in water but soluble in many organic solvents. They are typically divided into at least three classes: (1) "simple lipids," which include oils and waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0046] As used herein, the term "LNP" refers to lipid-nucleic acid particles or nucleic acid-lipid particles (e.g., stable nucleic acid-lipid particles). LNP refers to particles made of lipids (e.g., ionizable lipids of the present invention, non-cationic lipids, and conjugated lipids that prevent particle aggregation) and nucleic acids, where the nucleic acids (e.g., siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, mRNA, self-amplifying RNA, or plasmids containing plasmids from which interfering RNA or mRNA is transcribed) are encapsulated within the lipids. In one embodiment, the nucleic acids are at least 50% encapsulated within the lipids, in one embodiment, at least 75% encapsulated within the lipids, in one embodiment, at least 90% encapsulated within the lipids, and in one embodiment, the nucleic acids are completely encapsulated within the lipids. LNPs typically contain cationic lipids, non-cationic lipids, and lipid conjugates (e.g., PEG-lipid conjugates). LNPs are extremely useful for systemic applications because they can exhibit long circulatory lifetimes after intravenous (iv) injection, they can accumulate at distal sites (e.g., sites physically separated from the site of administration), and they can mediate transfected gene expression or silencing of target gene expression at these distal sites.

[0047] Lipid particles (e.g., LNPs) of the present invention typically have an average diameter of about 40 nm to about 150 nm. In one embodiment, lipid particles of the present invention have an average diameter of about 40 nm to about 80 nm. In one embodiment, lipid particles of the present invention have an average diameter of about 40 nm to about 70 nm. In one embodiment, lipid particles of the present invention have an average diameter of about 40 nm to about 60 nm. In one embodiment, lipid particles of the present invention have an average diameter of about 50 nm to about 60 nm. In one embodiment, lipid particles of the present invention have an average diameter of about 50 nm to about 150 nm. In one embodiment, lipid particles of the present invention have an average diameter of about 60 nm to about 130 nm. In one embodiment, lipid particles of the present invention have an average diameter of about 70 nm to about 110 nm. In one embodiment, lipid particles of the present invention have an average diameter of about 70 nm to about 90 nm. Lipid particles of the present invention are substantially non-toxic. Furthermore, nucleic acids, when present in lipid particles of the present invention, are resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in US Patent Publication Nos. 20040142025 and 20070042031, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0048] As used herein, "lipid-encapsulated" can refer to lipid particles that provide an active agent or therapeutic agent, such as a nucleic acid (e.g., an interfering RNA or 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).

[0049] The term "lipid conjugate" refers to a polymer-lipid conjugate that inhibits lipid particle aggregation. Such lipid conjugates include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), polysarcosine-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, PEG-lipid conjugates such as PEG conjugated to dialkyloxypropyl, diacylglycerol, cholesterol, phosphatidylethanolamine, and ceramide (see, e.g., U.S. Pat. No. 5,885,613, the disclosure of which is incorporated herein by reference in its entirety for all purposes), cationic PEG-lipids, and mixtures thereof. PEG can be directly conjugated to the lipid or can be linked to the lipid via a linker moiety. For example, any linker moiety suitable for coupling PEG to lipids can be used, including ester-free and ester-containing linker moieties. In a preferred embodiment, an ester-free linker moiety is used.

[0050] The term "amphipathic lipid" refers, in part, to any suitable lipid material in which the hydrophobic portion of the lipid material orients toward the hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Hydrophilic properties result 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 nonpolar groups, including, but not limited to, saturated and unsaturated long-chain aliphatic hydrocarbon groups, and those in which such groups are substituted with one or more aromatic, alicyclic, or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0051] 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 family, diacylglycerol, and β-acyloxyacid, also fall into the group of amphipathic lipids.In addition, the above amphipathic lipids can be mixed with other lipids, such as triglycerides and sterols.

[0052] The term "neutral lipid" refers to any of several lipid species that exist in either uncharged or neutral zwitterionic form at selected pHs. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0053] The term "non-cationic lipid" refers to any amphipathic lipid and any other neutral or anionic lipid.

[0054] 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.

[0055] The term "cationic lipid" refers to lipids that can be positively charged. These lipids include lipids that have one or more positively charged amine groups. Certain cationic lipids can be ionized (referred to as "ionizable lipids") and may exist in a charge-neutral or positively charged state depending on the surrounding pH.

[0056] In one embodiment, the cationic lipid is a compound of Formula (I) described herein, which in certain embodiments has a clearance rate such that less than 10% of the ionizable parent lipid remains 7 days after administration (e.g., in the assay described in Example 33).

[0057] The term "hydrophobic lipid" refers to a compound having a non-polar group, including but not limited to saturated and unsaturated long-chain aliphatic hydrocarbon groups, and such groups optionally substituted with one or more aromatic, alicyclic, or heterocyclic groups.Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0058] The term "fusogenic" refers to the ability of a lipid particle, such as an LNP, to fuse with a cell's membrane. The membrane can be either the plasma membrane or a membrane surrounding an organelle, e.g., an endosome, a nucleus, etc.

[0059] As used herein, the term "aqueous solution" refers to a composition that is composed entirely or partially of water.

[0060] As used herein, the term "organic lipid solution" refers to a composition composed entirely or partially of an organic solvent with a lipid.

[0061] As used herein, "distal site" refers to a physically separated site, and is not limited to adjacent capillary beds, but includes sites distributed widely throughout the organism.

[0062] "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 would significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0063] As used herein, "systemic delivery" refers to the 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 drug, while others cannot. Systemic delivery means that a useful amount, preferably a therapeutic amount, of the drug is exposed to most of the body. To achieve widespread biodistribution, a blood lifespan is generally required so that the drug does not undergo rapid degradation or clearance (such as by first-pass organs (liver, lung, etc.) or rapid non-specific cellular binding) before reaching the disease site 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.

[0064] As used herein, " local delivery " refers to the direct delivery of active agent or therapeutic agent, such as interfering RNA or mRNA, to target site in vivo.For example, drug can be delivered locally by direct injection to disease site (such as tumor) or other target site (such as inflammation site), or target organ (such as liver, heart, pancreas, kidney, etc.).

[0065] The term "mammal" refers to any mammalian species, for example, humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, farm animals, etc.

[0066] The term "cancer" refers to any member of a class of diseases characterized by the uncontrolled growth of abnormal cells. This term includes all known cancers and neoplastic diseases, whether malignant, benign, soft tissue, or solid, and all stages and grades of cancer, including pre- and post-metastatic. Examples of different types of cancer include, but are not limited to, lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, stomach 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), central nervous system cancer, 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 several other non-hepatic cancer cell types), and hepatoblastoma. As used herein, a "tumor" comprises one or more cancerous cells.

[0067] The term "anion precursor group" includes groups capable of forming ions at physiological pH. For example, the term includes the groups -COH, -OP(=O)(OH), -OS(=O)(OH), -OS(=O)(OH), and -B(OH). In one embodiment, the anion precursor is -COH.

[0068] In certain embodiments, PEG-C-DMA has the following structure: [ka] In the formula, n is selected so that the resulting polymer chain has a molecular weight of about 500 to about 10,000. In one embodiment, n is selected so that the resulting polymer chain has a molecular weight of about 500 to about 7500. In one embodiment, n is selected so that the resulting polymer chain has a molecular weight of about 500 to about 6000. In one embodiment, n is selected so that the resulting polymer chain has a molecular weight of about 500 to about 5000. In one embodiment, n is selected so that the resulting polymer chain has a molecular weight of about 500 to about 3000. In one embodiment, n is selected so that the resulting polymer chain has a molecular weight of about 1000 to about 7500. In one embodiment, n is selected so that the resulting polymer chain has a molecular weight of about 1000 to about 6000. In one embodiment, n is selected so that the resulting polymer chain has a molecular weight of about 1000 to about 5000. In one embodiment, n is selected so that the resulting polymer chain has a molecular weight of about 1000 to about 3000. In another embodiment, n is selected so that the resulting polymer chain has a molecular weight of about 2000. PEG-C-DMA can be prepared as described by 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.

[0069] Description of the embodiment 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).

[0070] In one aspect, the present invention provides lipid particles comprising: (a) one or more active or therapeutic agents; (b) one or more ionizable lipids of the present invention comprising from about 30 mol% to about 85 mol% of the total lipid present in the particle; (c) one or more non-cationic lipids comprising 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, comprising from 0.1 mol% to about 10 mol% of the total lipid present in the particle.

[0071] In one aspect, the present invention provides lipid particles comprising: (a) one or more active or therapeutic agents; (b) one or more ionizable lipids of the present invention comprising from about 50 mol% to about 85 mol% of the total lipid present in the particle; (c) one or more non-cationic lipids comprising 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, comprising from 0.5 mol% to about 2 mol% of the total lipid present in the particle.

[0072] 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 other certain embodiments, the lipid particle is substantially non-toxic to mammals, such as humans.

[0073] 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, an siRNA, an aiRNA, an miRNA, or a mixture thereof. In other certain 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.

[0074] 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 other certain 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.

[0075] In one embodiment, the active agent 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 molecules of the present invention are capable of silencing expression of a target sequence in vitro and / or in vivo.

[0076] 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.

[0077] In other embodiments, siRNA molecules comprise 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, siRNA comprises 2'OMe nucleotides (e.g., 2'OMe purine and / or pyrimidine nucleotides), such as 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, 2'OMe-cytosine nucleotides, and mixtures thereof. In certain cases, siRNA does not comprise 2'OMe-cytosine nucleotides. In other embodiments, siRNA comprises a hairpin loop structure.

[0078] siRNAs 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 nucleotides and / or guanosine nucleotides are modified at selective positions in the double-stranded region of the siRNA duplex. Regarding uridine nucleotide modifications, at least 1, 2, 3, 4, 5, 6, or more uridine nucleotides in the sense strand and / or antisense strand can be modified uridine nucleotides, such as 2'OMe-uridine nucleotides. In some embodiments, all uridine nucleotides in the sense strand and / or antisense strand are 2'OMe-uridine nucleotides. Regarding guanosine nucleotide modifications, at least 1, 2, 3, 4, 5, 6, or more guanosine nucleotides in the sense strand and / or antisense strand can be modified guanosine nucleotides, such as 2'OMe-guanosine nucleotides. In some embodiments, all guanosine nucleotides in the sense strand and / or antisense strand are 2'OMe-guanosine nucleotides.

[0079] In certain embodiments, at least one, two, three, four, five, six, seven, or more 5'-GU-3' motifs in an siRNA sequence can 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 can be in the sense strand, the antisense strand, or both strands of the siRNA sequence. The 5'-GU-3' motifs can be adjacent to each other or, alternatively, separated by one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more nucleotides.

[0080] In some preferred embodiments, modified siRNA molecules are less immunostimulatory than corresponding unmodified siRNA sequences. In such embodiments, modified siRNA molecules with reduced immunostimulatory properties advantageously retain RNAi activity against target sequences. In another embodiment, the immunostimulatory properties of modified siRNA molecules and their ability to silence target gene expression can be balanced and 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, modified siRNAs are 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 corresponding unmodified siRNAs. It will be readily apparent to one skilled in the art that the immunostimulatory 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 an appropriate lipid-based delivery system (such as the LNP delivery system disclosed herein).

[0081] In certain embodiments, the modified siRNA molecule is50 (i.e., half-maximal inhibitory concentration) is at least 10-fold lower than that of the corresponding unmodified siRNA (i.e., the modified siRNA has an IC 50 is the IC of the corresponding unmodified siRNA 50 In other embodiments, the modified siRNA has an IC 50 In yet another embodiment, the modified siRNA has an IC 50 is at least two-fold lower than that of the corresponding unmodified siRNA. Using methods known to those skilled 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 skilled in the art that the values ​​can be readily determined.

[0082] 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 sequence.

[0083] In some embodiments, the siRNA molecule does not comprise phosphate backbone modifications, for example, in the sense strand and / or antisense strand of the double-stranded region.In other embodiments, the siRNA comprises 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 preferred embodiments, the siRNA does not comprise phosphate backbone modifications.

[0084] In a further embodiment, siRNA does not contain 2'-deoxynucleotide, for example, in the sense strand and / or antisense strand of the double-stranded region.In yet a further embodiment, siRNA does not contain 2'-deoxynucleotide, for example, in the sense strand and / or antisense strand of the double-stranded region.In a preferred embodiment, siRNA does not contain 2'-deoxynucleotide.

[0085] 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 other certain 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.

[0086] The siRNA molecules described herein may have 1, 2, 3, 4, or more nucleotide 3' overhangs on one or both sides of the double-stranded region, or may lack overhangs (i.e., have blunt ends) on one or both sides of the double-stranded region. Preferably, the siRNA has 2-nucleotide 3' overhangs on either side of the double-stranded region. In certain cases, the 3' overhang on the antisense strand is complementary to the target sequence, and the 3' overhang on the sense strand is complementary to the complementary strand of the target sequence. Alternatively, the 3' overhang is not complementary to either the target sequence or its complementary strand. In some embodiments, the 3' overhang 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 (for example, 2'OMe purine and / or pyrimidine nucleotides), such as 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, 2'OMe-cytosine nucleotides, and mixtures thereof.

[0087] The siRNA may comprise at least one or a cocktail (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) of unmodified and / or modified siRNA sequences that silence 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, one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) modified siRNAs that silence target gene expression are present in the cocktail. In other certain cases, one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) unmodified siRNA sequences that silence target gene expression are present in the cocktail.

[0088] In some embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence or a portion thereof. In other embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that is 100% complementary to the target sequence or a portion thereof. In further embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that specifically hybridizes to the target sequence or a portion thereof.

[0089] 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, hi 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.

[0090] In some embodiments, the ionizable lipids of the present invention may comprise 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.

[0091] In some embodiments, the ionizable lipids of the present invention may comprise 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.

[0092] In other embodiments, the ionizable lipids of the present invention may comprise 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.

[0093] In yet other embodiments, the ionizable lipids of the present invention may comprise 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.

[0094] In still other embodiments, the ionizable lipids of the present invention may comprise 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.

[0095] In further embodiments, the ionizable lipids of the present invention may comprise 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.

[0096] In additional embodiments, the ionizable lipids of the present invention 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.

[0097] In the lipid particles of the present invention, the non-cationic lipid may comprise, for example, one or more anionic lipids and / or neutral lipids. In a preferred embodiment, the non-cationic lipid comprises 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.

[0098] 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.

[0099] 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.

[0100] In some embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) may comprise about 10 mol% to about 70 mol%, about 15 mol% to about 70 mol%, about 20 mol% to about 70 mol%, about 25 mol% to about 70 mol%, about 30 mol% to about 70 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.

[0101] In other embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) may comprise 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.

[0102] In yet other embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) may comprise from about 10 mol% to about 45 mol%, from about 13 mol% to about 45 mol%, from about 15 mol% to about 45 mol%, from about 20 mol% to about 45 mol%, from about 25 mol% to about 45 mol%, from about 30 mol% to about 45 mol%, or from about 35 mol% to about 45 mol% of the total lipid present in the particle.

[0103] In still other embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) may comprise from about 10 mol% to about 40 mol%, from about 13 mol% to about 40 mol%, from about 15 mol% to about 40 mol%, from about 20 mol% to about 40 mol%, from about 25 mol% to about 40 mol%, or from about 30 mol% to about 40 mol% of the total lipid present in the particle.

[0104] In further embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) may comprise from about 10 mol% to about 35 mol%, from about 13 mol% to about 35 mol%, from about 15 mol% to about 35 mol%, from about 20 mol% to about 35 mol%, or from about 25 mol% to about 35 mol% of the total lipid present in the particle.

[0105] In still further embodiments, the non-cationic lipid (e.g., one or more phospholipids and / or cholesterol) may comprise from about 10 mol% to about 30 mol%, from about 13 mol% to about 30 mol%, from about 15 mol% to about 30 mol%, from about 20 mol% to about 30 mol%, from about 10 mol% to about 25 mol%, from about 13 mol% to about 25 mol%, or from about 15 mol% to about 25 mol% of the total lipid present in the particle.

[0106] 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.

[0107] 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.

[0108] 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 thereof 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 or range therein) of cholesterol or a derivative thereof.

[0109] In a more 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% of DPPC and about 20 mol% of 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 or range therein) of phospholipids of the total lipid present in the particle, and and (ii) about 10 mol% to about 30 mol%, about 10 mol% to about 25 mol%, about 10 mol% to about 20 mol%, about 15 mol% to about 30 mol%, about 20 mol% to about 30 mol%, about 15 mol% to about 25 mol%, about 12 mol% to about 28 mol%, about 14 mol% to about 26 mol%, or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mol% (or any fraction or range therein) of cholesterol or a derivative thereof of the total lipid present in the particle.

[0110] complex lipid 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 a mixture thereof. The PEG-DAA conjugate may be PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmityloxypropyl (C16), PEG-distearyloxypropyl (C18), or a mixture thereof.

[0111] Additional 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. Further additional PEG-lipid conjugates suitable for use in the present invention include, but are not limited to, 1-[8'-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3',6'-dioxaoctanyl]carbamoyl-w-methyl-poly(ethylene glycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S. Patent No. 7,404,969, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0112] 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.

[0113] In certain cases, conjugated lipids (e.g., PEG-lipid conjugates) may comprise 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., 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 or range therein) of the total lipid present in the particle.

[0114] In the lipid particles of the present invention, active agent or therapeutic agent is completely encapsulated in the lipid portion of particle, thereby protecting active agent or therapeutic agent from nuclease degradation.In a preferred embodiment, LNPs containing nucleic acid such as interfering RNA (for example, siRNA) or mRNA are completely encapsulated in the lipid portion of particle, thereby protecting nucleic acid from nuclease degradation.In certain cases, the nucleic acid in LNP is not substantially decomposed after particle is exposed to nuclease at 37 ℃ for at least about 20, 30, 45 or 60 minutes.In other specific cases, the nucleic acid in LNP is not substantially decomposed after particle is incubated in serum at 37 ℃ 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 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.

[0115] The term "fully encapsulated" indicates that the active agent or therapeutic agent in the lipid particle is not significantly degraded after exposure to serum or nuclease or protease assays, which can significantly degrade free DNA, RNA, or protein. In a fully encapsulated system, preferably less than about 25% of the active agent or therapeutic agent in the particle is degraded, more preferably less than about 10%, and most preferably less than about 5% of the active agent or therapeutic agent in the particle is degraded in a process that would normally degrade 100% of the free active agent or therapeutic agent. In the context of nucleic acid therapeutic agents, complete encapsulation can be determined by the 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 particle is stable in serum, i.e., it is not rapidly degraded into its constituent components upon in vivo administration.

[0116] In another aspect, the present invention provides a lipid particle (e.g., LNP) composition comprising a plurality of lipid particles. In a preferred embodiment, the 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%, or about 40% to about 90% 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 fully encapsulated within the lipid portion of the lipid particle (e.g., LNP) having encapsulated therein.

[0117] Typically, lipid particles (e.g., LNPs) of the present invention have a lipid:active agent (e.g., lipid:nucleic acid) ratio (mass / mass) of about 1 to about 100. In some cases, the lipid:active agent (e.g., lipid:nucleic acid) ratio (mass / mass) 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 present invention have a lipid:active agent (e.g., lipid:nucleic acid) ratio (mass / mass) of about 5 to about 15, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (or any fraction thereof or range therein).

[0118] Typically, the lipid particles (e.g., LNP) of the present invention have an average diameter of about 40 nm to about 150 nm. In a preferred embodiment, the lipid particles (e.g., LNP) of the present invention have an average diameter of about 40 nm to about 130 nm, about 40 nm to about 120 nm, about 40 nm to about 100 nm, about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 40 nm to about 80 nm, about 40 nm to about 60 nm, about 50 nm to about 60 nm, about 60 nm to about 120 nm, about 60 nm to about 110 nm, or about 60 nm to about 1 00 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 150 nm, 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm (or any fraction thereof or range therein).

[0119] In one specific embodiment of the present invention, the LNP comprises: (a) one or more unmodified and / or modified nucleic acid molecules (e.g., interfering RNAs that silence expression of target genes, such as siRNA, aiRNA, or miRNA; or mRNA that drives expression of a target protein); (b) an ionizable lipid of the present invention, comprising about 56.5 mol% to about 66.5 mol% of the total lipids present in the particle; (c) a non-cationic lipid, comprising about 31.5 mol% to about 42.5 mol% of the total lipids present in the particle; and (d) a complex lipid that inhibits particle aggregation, comprising about 1 mol% to about 2 mol% of the total lipids present in the particle. This specific embodiment of the LNP is generally referred to herein as a "1:62" formulation.

[0120] In another specific embodiment of the present 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, aiRNA, or miRNA, or an mRNA that drives expression of a target protein); (b) an ionizable lipid of the present invention, comprising about 52 mol% to about 62 mol% of the total lipid present in the particle; (c) a non-cationic lipid, comprising about 36 mol% to about 47 mol% of the total lipid present in the particle; and (d) a complex lipid that inhibits particle aggregation, comprising about 1 mol% to about 2 mol% of the total lipid present in the particle. This specific embodiment of the LNP is generally referred to herein as a "1:57" formulation.

[0121] The present invention also provides pharmaceutical compositions comprising a lipid particle (eg, LNP) described herein and a pharmaceutically acceptable carrier.

[0122] In a further aspect, the present invention provides a method for introducing one or more active or therapeutic agents (e.g., nucleic acids) into cells, comprising contacting the cells with lipid particles (e.g., LNPs) described herein. In one embodiment, the cells are in a mammal, and the mammal is a human. In another embodiment, the present invention provides a method for in vivo delivery of one or more active or therapeutic agents (e.g., nucleic acids), comprising administering lipid particles (e.g., LNPs) described herein to a mammalian subject. In preferred embodiments, modes of administration include, but are not limited to, oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, and intradermal. Preferably, the mammalian subject is a human.

[0123] In one embodiment, at least about 5%, 10%, 15%, 20% or 25% of the total injected dose of lipid particles (e.g., LNP) is present in plasma about 8, 12, 24, 36 or 48 hours after injection.In other embodiments, about 8, 12, 24, 36 or 48 hours after injection, 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., LNP) is present in plasma.In certain cases, about 1 hour after administration, more than about 10% of the particles are present in the plasma of a mammal.In other certain cases, the presence of lipid particles (e.g., LNP) can be detected at least about 1 hour after administration of the particles. In certain embodiments, the presence of an active or therapeutic agent, such as an interfering RNA (e.g., siRNA) or mRNA, is detectable in cells (e.g., lung, liver, tumor, or site of inflammation) about 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, or 96 hours after administration. In other embodiments, downregulation of target sequence expression by an active or therapeutic agent, such as an interfering RNA (e.g., siRNA), is detectable about 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, or 96 hours after administration. In yet other embodiments, downregulation of target sequence expression by an active or therapeutic agent, such as an interfering RNA (e.g., siRNA), 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 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, 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 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 or therapeutic agent, such as an mRNA or self-amplifying RNA, occurs preferentially in tumor cells or cells at a site 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 a site proximal or distal to the site of administration or in cells of the lung, liver, or tumor is detectable about 12 hours, 24 hours, 48 ​​hours, 72 hours, or 96 hours, or about 6 days, 8 days, 10 days, 12 days, 14 days, 16 days, 18 days, 19 days, 20 days, 22 days, 24 days, 26 days, or 28 days after administration. In additional embodiments, the lipid particles (e.g., LNPs) of the present invention are administered parenterally or intraperitoneally.

[0124] 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 comprising an interfering RNA sequence (e.g., siRNA). 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., siRNA) to the liver and / or tumor of a mammalian subject. In certain embodiments, the disease or disorder is associated with gene expression and / or overexpression, and the gene expression or overexpression is reduced by the interfering RNA (e.g., siRNA). In other specific 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., siRNA) is 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 (e.g., siRNA) is delivered in vitro (e.g., using LNPs described herein), and the cells are re-infused into the patient.

[0125] In additional aspects, the present invention provides lipid particles (e.g., LNPs) comprising asymmetric interfering RNA (aiRNA) molecules that silence the expression of target genes, and methods of using such particles to silence the expression of target genes.

[0126] In one embodiment, the aiRNA molecule comprises a double-stranded (duplex) region of about 10 to about 25 (base pairs) 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.

[0127] In certain cases, the aiRNA molecule comprises a double-stranded (duplex) region approximately 12-20, 12-19, 12-18, 13-17, or 14-17 nucleotides in length, more usually 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In other certain cases, the 5' and 3' overhangs on the antisense strand comprise sequences 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.

[0128] In other embodiments, the aiRNA molecule comprises modified nucleotides selected from the group consisting of 2'OMe nucleotides, 2'F nucleotides, 2'-deoxynucleotides, 2'-O-MOE nucleotides, LNA nucleotides, and mixtures thereof. In preferred embodiments, the aiRNA 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.

[0129] In a related aspect, the present invention provides lipid particles (e.g., LNPs) comprising microRNA (miRNA) molecules that silence the expression of target genes, and methods of using such compositions to silence target gene expression.

[0130] In one embodiment, the miRNA molecule has a length of about 15 to about 60 nucleotides, and the miRNA molecule is capable of silencing expression of a target gene.

[0131] In certain cases, the miRNA molecule is about 15-50, 15-40, or 15-30 nucleotides in length, more usually 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.

[0132] In some embodiments, the miRNA molecule comprises modified nucleotides selected from the group consisting of 2'OMe nucleotides, 2'F nucleotides, 2'-deoxynucleotides, 2'-O-MOE nucleotides, LNA nucleotides, and mixtures thereof. In 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.

[0133] 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) through the expression of one or more target proteins. As a non-limiting example, the methods of the present invention are useful for the in vivo delivery of one or more mRNA molecules to mammalian subjects. In other specific embodiments, a therapeutically effective amount of lipid particles (e.g., LNPs) can be administered to a mammal. In some cases, one or more mRNA molecules are formulated into LNPs, and the particles are administered to a patient in need of such treatment. In other cases, cells are removed from a patient, one or more mRNA molecules are delivered in vitro (e.g., using LNPs described herein), and the cells are re-infused into the patient.

[0134] 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; guide RNA; self-amplifying RNA; circular RNA; DNA, including plasmid DNA and closed-end DNA), 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.

[0135] activator Active agents (e.g., therapeutic agents) include any molecule or compound capable of exerting a desired effect in 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-inducing factors, 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 pharmaceutical or drug known to those of skill in the art.

[0136] 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, and in another embodiment, the therapeutic agent derivative is a prodrug that lacks therapeutic activity but becomes active upon further modification.

[0137] nucleic acid In certain embodiments, 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, oligonucleotides of the present invention are about 15 to about 60 nucleotides in length. Nucleic acids may 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.

[0138] In the context of this 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, or portions thereof, containing non-naturally occurring monomers that 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.

[0139] Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleotides. Deoxyribooligonucleotides consist of a five-carbon sugar called deoxyribose covalently linked to phosphate at the 5' and 3' carbons of the sugar, forming an unbranched alternating polymer. Ribooligonucleotides consist of a similar repeating structure in which the five-carbon sugar is ribose.

[0140] The nucleic acid present in the lipid-nucleic acid particle of the present invention includes any known form of nucleic acid.The nucleic acid used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrid.Examples of double-stranded DNA are described herein, including, for example, structural genes, genes containing regulatory and termination regions, and self-replicating systems such as viral DNA or plasmid DNA.Examples of double-stranded RNA are described herein, including, for example, siRNA, and other RNAi agents such as aiRNA and pre-miRNA.Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, mature miRNAs, and triplex-forming oligonucleotides.

[0141] Nucleic acids of the present invention can be of various lengths, generally depending on the particular form of the nucleic acid. For example, in certain embodiments, a plasmid or gene can be from about 1,000 to about 100,000 nucleotide residues in length. In certain embodiments, an oligonucleotide can range from about 10 to about 100 nucleotides in length. In various related embodiments, the oligonucleotide, whether single-stranded, double-stranded, or triple-stranded, can range in length from about 10 to about 60 nucleotides, about 15 to about 60 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 30 nucleotides.

[0142] In certain embodiments, the oligonucleotides (or strands thereof) of the present invention specifically hybridize to or are complementary to a target polynucleotide sequence. As used herein, the terms "specifically hybridizable" and "complementary" refer to a sufficient degree of complementarity such that stable and specific binding occurs between the target DNA or RNA and the oligonucleotide. It should be understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. In preferred embodiments, an oligonucleotide is specifically hybridizable if its binding to the target sequence interferes with the normal function of the target sequence, causing loss of utility or expression from the target sequence, and if there is sufficient complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, or under the conditions in which the assay is performed in the case of in vitro assays. 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.

[0143] 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 in length, preferably about 15 to about 30 nucleotides in length. In certain embodiments, the siRNA contains at least one modified nucleotide. Modified siRNAs are generally less immunostimulatory than the corresponding unmodified siRNA sequence and retain RNAi activity against the target gene of interest. In some embodiments, the modified siRNA contains at least one 2'OMe purine or 2'OMe 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).

[0144] Modified siRNAs generally comprise 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. 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.

[0145] 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.

[0146] 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.) of the nucleotides in the double-stranded region of the siRNA or about 1% to about 20% (e.g., about 1% to 20%) of the nucleotides in the double-stranded region of the siRNA. %, 2%~20%, 3%~20%, 4%~20%, 5%~20%, 6%~20%, 7%~20%, 8%~20%, 9%~20%, 10%~20%, 11%~20%, 12%~20%, 13%~20%, 14%~20%, 15%~20%, 16%~20%, 17%~20%, 18 %~20%, 19%~20%, 1%~19%, 2%~19%, 3%~19%, 4%~19%, 5%~19%, 6%~19%, 7%~19%, 8%~19%, 9%~19%, 10%~19%, 11%~19%, 12%~19%, 13%~19%, 14%~19%, 15%~19%, 16%~19%, 17%~19%, 18%~19%, 1%~18%, 2%~18%, 3%~18%, 4%~18%, 5%~18%, 6%~18%, 7%~18%, 8%~18%, 9%~18%, 10%~18%, 11%~18%, 12%~18%, 13%~18%, 14%~18% 8%, 15%~18%, 16%~18%, 17%~18%, 1%~17%, 2%~17%, 3%~17%, 4%~17%, 5%~17%, 6%~17%, 7%~17%, 8%~17%, 9%~17%, 10%~17%, 11%~17%, 12%~17%, 13%~17%, 14% ~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.

[0147] In further embodiments, for example, when one or both strands of an 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%. less than, or 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%, or 29% to 30% modified nucleotides).

[0148] Selection of siRNA sequences Suitable siRNA sequence can be identified by any means known in the art.Usually, the method described in Elbashir et al., Nature, 411:494-498(2001) and Elbashir et al., EMBO J., 20:6877-6888(2001) is combined with the rational design rule described in Reynolds et al., Nature Biotech., 22(3):326-330(2004).

[0149] 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, for example, Elbashir et al., EMBO J., 20:6877-6888 (2001)). The nucleotides adjacent to the 3' of the dinucleotide sequence are identified as potential siRNA sequences (i.e., target sequences or sense strand sequences). Usually, 19, 21, 23, 25, 27, 29, 31, 33, 35, or more nucleotides adjacent to the 3' of the dinucleotide sequence are identified as potential siRNA sequences. In some embodiments, the dinucleotide sequence is an AA or NA sequence, and 19 nucleotides adjacent to the 3' of the AA or NA dinucleotide are identified as potential siRNA sequences. The siRNA sequences are usually spaced at different positions along the length of the target gene. To further enhance the silencing efficiency of siRNA sequences, potential siRNA sequences may be analyzed to identify regions that do not contain homologous regions with other coding sequences, for example, in the target cell or organism. For example, a suitable siRNA sequence of approximately 21 base pairs typically does not have more than 16-17 consecutive base pairs that are homologous to coding sequences in the target cell or organism. When the siRNA sequence is expressed from an RNA Pol III promoter, an siRNA sequence lacking more than four consecutive A's or T's is selected.

[0150] Once potential siRNA sequences are identified, complementary sequences (i.e., antisense strand sequences) can be designed. Potential siRNA sequences can also be analyzed using various criteria known in the art. For example, to improve their silencing efficiency, siRNA sequences can be analyzed using rational design algorithms to identify sequences with one or more of the following characteristics: (1) a G / C content of about 25% to about 60%, (2) at least three A / Us at positions 15 to 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) a non-G / C at position 19 of the sense strand, and (8) a non-G at position 13 of the sense strand. An siRNA design tool incorporating an algorithm that assigns appropriate values ​​to each of these characteristics and is useful for selecting siRNAs can be found, for example, at http: / / boz094.ust.hk / RNAi / siRNA. One skilled in the art will appreciate that sequences possessing one or more of the aforementioned properties may be selected for further analysis and testing as potential siRNA sequences.

[0151] Furthermore, potential siRNA sequences that have one or more of the following criteria can often be excluded as siRNAs: (1) sequences containing regions with four or more identical bases, (2) sequences containing homopolymers of G (i.e., to reduce the possibility of non-specific effects due to the structural properties of these polymers), (3) sequences containing triple-base motifs (e.g., GGG, CCC, AAA, or TTT), (4) sequences containing regions with seven or more G / Cs, and (5) sequences containing direct repeats of four or more bases within the candidate, resulting in an internal foldback structure. However, those skilled in the art will understand that sequences with one or more of the above-mentioned characteristics can still be selected as potential siRNA sequences for further analysis and testing.

[0152] 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 the secondary structure at target site, for example, as described in Luo et al., Biophys.Res.Commun., 318:303-310 (2004).For example, the secondary structure at target site can be modeled using the Mfold algorithm (available at http: / / www.bioinfo.rpi.edu / applications / mfold / RNA / form1.cgi), and the siRNA sequence that has low base pairing and stem-loop type secondary structure and favors accessibility at target site can be selected.

[0153] After a potential siRNA sequence is identified, the sequence can be analyzed for the presence of any immunostimulatory properties, for example, using an in vitro cytokine assay or an in vivo animal model. Motifs within the sense and / or antisense strands of the siRNA sequence, such as GU-rich motifs (e.g., 5'-GU-3', 5'-UGU-3', 5'-GUGU-3', 5'-UGUGU-3', etc.), can also provide an indication of whether the sequence may be immunostimulatory. If an siRNA molecule is found to be immunostimulatory, the siRNA molecule can then be modified to reduce its immunostimulatory properties as described herein. As a non-limiting example, to determine whether an siRNA is an immunostimulatory or non-immunostimulatory siRNA, the siRNA sequence can be contacted with mammalian responder cells under conditions such that the cells produce a detectable immune response. The mammalian responder cells can be 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 may 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. An siRNA molecule identified as immunostimulatory can then be modified to reduce its immunostimulatory properties by substituting at least one of the nucleotides in the sense and / or antisense strands with a modified nucleotide. 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 substituted 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 have been reduced or suppressed.

[0154] 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), the monoclonal-polyclonal antibody sandwich assay (Wide et al., in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970)), the "Western blot" method of Gordon et al. (U.S. 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 including the use of fluorescent dyes (Brooks et al., Clin. Exp. Immunol., 39:477 (1980); and neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad. Sci. USA, 81:2396-2400 (1984)). In addition to the above immunoassays, many 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.

[0155] Non-limiting examples of in vivo models for detecting immune responses include, for example, the in vivo mouse cytokine induction assay described in Judge et al., Mol. Ther., 13:494-505 (2006). In certain embodiments, the assay can be performed as follows: (1) siRNA can be administered via standard intravenous injection into the lateral tail vein; (2) blood can be collected by cardiac puncture approximately 6 hours after administration and processed as plasma for cytokine analysis; and (3) cytokines can be quantified using 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.)).

[0156] Monoclonal antibodies that specifically bind to cytokines and growth factors are commercially available from multiple 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)). The production of monoclonal antibodies has been previously described and can be performed by any means known in the art (Buhring et al., in Hybridoma, Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, monoclonal antibodies are labeled for easy detection (e.g., with any composition detectable by spectroscopic, photochemical, biochemical, electrical, optical, or chemical means).

[0157] Creation of siRNA molecules siRNA can be provided in several forms, for example, as one or more isolated small interfering RNA (siRNA) duplexes, as longer double-stranded RNA (dsRNA), or as the siRNA or dsRNA transcribed from the transcription cassette in DNA plasmid.SiRNA sequence can have overhang (for example, 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 end).

[0158] RNA population can be used to obtain long precursor RNA, or long precursor RNA with substantial or complete identity to selected target sequence can be used to make siRNA.RNA can be isolated from cells or tissue, 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 tissue, transcribed from cDNA, subtracted, selected, etc.), or can represent a single target sequence.RNA can be naturally occurring (for example, separated from tissue or cell sample), synthesized in vitro (for example, using T7 or SP6 polymerase and PCR product or cloned cDNA), or chemically synthesized.

[0159] In order to form long dsRNA, in the case of synthetic RNA, complement is also transcribed in vitro and hybridized to form dsRNA.When using naturally occurring RNA population, for example, by transcribing the cDNA corresponding to RNA population or using RNA polymerase, RNA complement is also provided (for example, to form dsRNA for E. coli RNAse III or Dicer digestion).Then, precursor RNA is hybridized to form double-stranded RNA that is digested.dsRNA can be directly administered to subject, or can be digested in vitro before administration.

[0160] 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 are PCR techniques (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. Further basic documents disclosing general methods of use in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989), Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990), and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994), the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0161] Preferably, siRNA is chemically synthesized.The oligonucleotides comprising the siRNA molecules of the present invention can be synthesized using any of a variety of techniques known in the art, such as those described in Usman et al., J.Am.Chem.Soc.,109:7845(1987), Scaringe et al., Nucl.Acids Res.,18:5433(1990), Wincott et al., Nucl.Acids Res.,23:2677-2684(1995), and Wincott et al., Methods Mol.Bio.,74:59(1997).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, 0.2 μmol scale syntheses can be performed on a 96-well plate synthesizer from Protogene (Palo Alto, Calif.), although larger or smaller scale syntheses are also within the scope of the present invention. Suitable reagents for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those of skill in the art.

[0162] siRNA molecules can also be synthesized by tandem synthesis techniques, in which both strands are synthesized as a single continuous oligonucleotide fragment or strand separated by a cleavable linker, which is then cleaved to obtain separate fragments or strands that hybridize to form an siRNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. Tandem siRNA synthesis 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 constructed from two separate oligonucleotides, where one oligonucleotide contains the sense strand of the siRNA and the other contains the antisense strand. For example, each strand can be synthesized separately and then linked together by hybridization or ligation after synthesis and / or deprotection. In other specific 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.

[0163] siRNA sequence modification In certain embodiments, the siRNA molecule comprises a duplex having two strands and at least one modified nucleotide in the double-stranded region, wherein each strand is about 15 to about 60 nucleotides in length. Advantageously, the modified siRNA is less immunostimulatory than the corresponding unmodified siRNA sequence, while retaining 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 select uridine and / or guanosine nucleotides within the siRNA duplex to eliminate immune responses caused by the siRNA while retaining its ability to silence target gene expression.

[0164] The modified nucleotide suitable for use in the present invention includes, but is 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 with both the Watson-Crick and Hoogsteen faces of complementary guanine nucleotides in a duplex (see, for example, 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 siRNA molecules.

[0165] In certain embodiments, the siRNA molecule may further comprise one or more chemical modifications, such as an end-capping moiety, a phosphate backbone modification, etc. Examples of end-capping 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, 3'-3'-inverted abasic moieties, 5'- ... These include, but are not limited to, '-5'-inverted deoxy abasic moieties, 5'-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 1,4-butanediol phosphate, 3'-phosphoramidate, 5'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 5'-amino, 3'-phosphorothioate, 5'-phosphorothioate, phosphorodithioate, and bridged or non-bridged 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 bond) include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal and alkylsilyl substitution (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 appear at the 5'-end and / or 3'-end of the sense strand, antisense strand, or both strands of siRNA. The disclosures of these references are incorporated herein by reference in their entireties for all purposes.

[0166] In some embodiments, the sense strand and / or antisense strand of the siRNA molecule can further comprise about one to about four (e.g., one, two, three, or four) 2'-deoxyribonucleotides and / or a 3'-end overhang having any combination of modified and unmodified nucleotides. Further examples of the types of modified nucleotides and chemical modifications that can be introduced into siRNA molecules are described, for example, in British Patent No. GB2,397,818B and U.S. Patent Publication Nos. 20040192626, 20050282188, and 20070135372, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0167] The siRNA molecules described herein can optionally contain one or more non-nucleotides in one or both strands of the siRNA.As used herein, the term "non-nucleotide" refers to any group or compound, including sugar and / or phosphate substitution, that can be incorporated into a nucleic acid strand in place of one or more nucleotide units, 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.

[0168] 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, for example, a biodegradable linker.The conjugate can also be bound to siRNA via, for example, a carbamate group or other linking group (see, for example, US Patent 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 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 U.S. 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, and cationic amino acid conjugate molecules described in U.S. Patent Publication No. 20050153337. Additional examples include hydrophobic groups, membrane active compounds, cell-permeable compounds, cell targeting signals, interaction modifiers, and steric stabilizer conjugate molecules described in U.S. Patent Publication No. 20040167090.Further examples include the 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 the improved pharmacokinetic profile, bioavailability and / or stability of siRNA while maintaining RNAi activity.Therefore, those skilled in the art can use various well-known in vitro cell cultures or in vivo animal models to screen the siRNA molecules that are conjugated with various conjugates, and identify those that have improved properties and full RNAi activity.The disclosures of the above-mentioned patent documents are incorporated herein by reference in their entirety for all purposes.

[0169] 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.The gene of interest includes, but is not limited to, genes related to viral infection and survival, genes related to metabolic diseases and disorders (e.g., liver disease and liver damage), genes related to tumorigenesis and cell transformation (e.g., cancer), angiogenesis genes, immunoregulatory genes such as those related to inflammatory and autoimmune responses, ligand receptor genes, and genes related to neurodegenerative disorders.In certain embodiments, the gene of interest is expressed in hepatocytes.

[0170] Genes associated with viral infection and survival include genes expressed by viruses to bind to, invade, and replicate within cells. 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 the irreplication, 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. 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)).

[0171] 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 numbers NC_002549, AY769362, NC_006432, NC_004161, AY729654, AY354458, AY142960, AB050936, AF522874, AF499101, AF272001, and AF086833. The sequence of Ebola virus VP24 is described, for example, in GenBank accession numbers U77385 and AY058897. The sequence of Ebola virus L-pol is described, for example, in Genbank accession number X67110. The sequence of Ebola virus VP40 is described, for example, in Genbank accession number AY058896. The sequence of Ebola virus NP is described, for example, in Genbank accession number AY058895. The sequence of Ebola virus GP is described, for example, in Genbank accession number AY058898, Sanchez et al., Virus Res., 29:215-240 (1993), Will et al., J. Virol., 67:1203-1210 (1993), Volchkov et al., FEBS Lett., 305:181-184 (1992), and U.S. Patent No. 6,713,069. Additional Ebola virus sequences are described, for example, in Genbank accession numbers L11365 and X61274. The complete genome sequence of Marburg virus is described, for example, in Genbank accession numbers NC_001608, AY430365, AY430366, and AY358025. The sequence of Marburg virus GP is described, for example, in Genbank accession numbers AF005734, AF005733, and AF005732. The sequence of Marburg virus VP35 is described, for example, in Genbank accession numbers AF005731 and AF005730.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 Publication No. 20070135370, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0172] 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 are, for example, those listed under Genbank accession numbers NC_004522, AY818138, AB166863, AB188817, AB189046, AB189054, AB189062, AY646169, AY646177, AY651486, AY651493, AY651494, AY651495, AY651496, AY6 51497, 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, under Genbank accession numbers AY818132, AY790280, AY646171, AY818132, AY818133, AY646179, AY818134, AY551934, AY651613, AY651610, AY651620, AY651617, AY651600, AY6 51611, 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 Publication No. 20070218122, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0173] 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, the 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 set forth, for example, in Genbank accession numbers NC_004102 (HCV genotype 1a), AJ238799 (HCV genotype 1b), NC_009823 (HCV genotype 2), NC_009824 (HCV genotype 3), NC_009825 (HCV genotype 4), NC_009826 (HCV genotype 5), and NC_009827 (HCV genotype 6). The nucleic acid sequence of hepatitis A virus is described, for example, in Genbank accession number NC_001489, the nucleic acid sequence of hepatitis B virus is described, for example, in Genbank accession number NC_003977, the nucleic acid sequence of hepatitis D virus is described, for example, in Genbank accession number NC_001653, the nucleic acid sequence of hepatitis E virus is described, for example, in Genbank accession number NC_001434, and the nucleic acid sequence of hepatitis G virus is described, for example, in Genbank accession number NC_001710. Silencing of sequences encoding genes associated with 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 targeting hepatitis virus nucleic acid sequences include those described in U.S. Patent Publication Nos. 20060281175, 20050058982, and 20070149470, U.S. Patent No. 7,348,314, and U.S. Provisional Patent Application No. 61 / 162,127, filed March 20, 2009, the disclosures of which are incorporated by reference in their entireties herein for all purposes.

[0174] 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 number NM_007121), farnesoid X receptor (FXR) (Genbank accession number NM_005123), sterol regulatory element binding protein (SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG coenzyme-A reductase), apolipoprotein B (ApoB) (Genbank accession number NM_000384), apolipoprotein CIII (ApoC3) (Genbank accession numbers NM_000040 and NG_008949)). REGION:5001.8164), and apolipoprotein E (ApoE) (Genbank accession numbers NM_000041 and NG_007084 REGION:5001.8612)), 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. (See, e.g., Lehmann et al., J. Biol. Chem., 272:3137-3140 (1997), Janowski et al., Nature, 383:728-731 (1996), and Peet et al., Cell, 93:693-704 (1998). Those skilled in the art will understand that genes associated with metabolic diseases and disorders (e.g., disorders in which the liver is a target, and liver diseases and disorders) include genes expressed in the liver itself as well as genes expressed in other organs and tissues.Silencing the sequence encoding the gene related to metabolic disease 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 gene include those described in US Patent Application 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 gene 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.

[0175] 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 number NM_004523); tyrosine kinases such as polo-like kinase 1 (PLK-1) (Genbank accession number NM_005030, Barr et al., Nat. Rev. Mol. Cell. Biol., 5:429-440 (2004) WEE1 (Genbank accession numbers NM_003390 and NM_001143976); inhibitors of apoptosis such as XIAP (Genbank accession number NM_001167); CSN1, CSN2, CSN3, CSN4, CSN5 (JAB1; Genbank accession number NM_006837), CSN6, CSN7A, These include COP9 signalosome subunits such as CSN7B and CSN8; ubiquitin ligases such as COP1 (RFWD2; Genbank accession numbers NM_022457 and NM_001001740); and histone deacetylases such as HDAC1, HDAC2 (Genbank accession number NM_001527), HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, and HDAC9. Non-limiting examples of siRNA molecules targeting IAP 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 PLK-1 genes include those described in U.S. Patent Publication Nos. 20050107316 and 20070265438, and U.S. Patent Application No. 12 / 343,342, filed December 23, 2008, the disclosures of which are incorporated herein by reference in their entirety for all purposes. Non-limiting examples of siRNA molecules targeting CSN5 genes include those described in U.S. Provisional Patent Application No. 61 / 045,251, filed April 15, 2008, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0176] Further examples of gene sequences associated with tumorigenesis and cell transformation include translocation sequences, e.g., MLL fusion genes, BCR-ABL (Wilda et al., Oncogene, 21:5716 (2002); Scherr et al., Blood, 101:1566 (2003)), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO, and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003)); overexpression sequences, e.g., multidrug resistance genes (Nieth et al., FEBS Lett., 545:144 (2003); Wu et al., Cancer Res., 63:1515 (2003)), cyclins (Li et al., Cancer Res., 63:3593 (2003); Zou et al. al., Genes Dev., 16:2923(2002)), beta-catenin (Verma et al., Clin Cancer Res., 9:1291(2003)), telomerase gene (Kosciolek et al., Mol Cancer Ther., 2:209(2003)), c-MYC, N-MYC, BCL-2, growth factor receptors (e.g., EGFR / ErbB1 (Genbank accession numbers NM_005228, NM_201282, NM_201283, and NM_201284, also Nagy et al. See, e.g., et al. Exp. Cell Res., 285: 39-49 (2003), including ErbB2 / HER-2 (Genbank accession number NM_004448 and NM_001005862), ErbB3 (Genbank accession number NM_001982 and NM_001005915), and ErbB4 (Genbank accession number NM_005235 and NM_001042599); and mutant sequences such as RAS (reviewed in Tuschl and Borkhardt, Mol. Interventions, 2: 158 (2002)).Non-limiting examples of siRNA molecules that target EGFR gene 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.

[0177] 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, such as integrins, selectins, and metalloproteinases. The aforementioned examples are not exhaustive. Those 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 can be included as a template sequence.

[0178] Angiogenesis gene can promote the formation of new blood vessels.Particularly of interest is vascular endothelial growth factor (VEGF) (Reich et al., Mol.Vis.,9:210(2003)) or VEGFR.The siRNA sequence of targeting VEGFR is described in, for example, GB2396864, US Patent Application Publication No. 20040142895 and CA2456444, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0179] Anti-angiogenic genes can inhibit neovascularization. These genes are particularly useful for treating cancer, in which angiogenesis plays a role in the pathological development of disease. Examples of anti-angiogenic genes include, but are not limited to, endostatin (see, for example, U.S. Patent No. 6,174,861), angiostatin (see, for example, U.S. Patent No. 5,639,725) and VEGFR2 (see, for example, Decaussin et al., J.Pathol., 188:369-377 (1999)), the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0180] 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 secondary 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.

[0181] 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 modulate (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)).

[0182] Other specific target genes that can be targeted by nucleic acids (e.g., by siRNA) to downregulate or silence gene expression include aortic smooth muscle alpha-2 actin (ACTA2), alcohol dehydrogenase 1A (ADH1A), alcohol dehydrogenase 4 (ADH4), alcohol dehydrogenase 6 (ADH6), afamin (AFM), angiotensinogen (AGT), serine-pyruvate aminotransferase (AGXT), alpha-2-H S-glycoprotein (AHSG), aldo-ketoreductase 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-100 (APOB), apolipoprotein C-IV (APOC4), apolipoprotein B-II (APOA2 ... 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), a 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 (C8 G), complement component C9 (C9), calmodulin-binding transcription activator 1 (CAMTA1), CD38 (CD38), complement factor B (CFB), complement factor H-related protein 1 (CFHR1), complement factor H-related protein 2 (CFHR2), complement factor H-related protein 3 (CFHR3), cannabinoid receptor 1 (CNR1), ceruloplasmin (CP), carboxypeptidase B2 (CPB2), connective tissue growth factor (CTGF), C-X-C motif chemokine 2 (CXCL2), cytochrome P450 1A2 (CYP1A2), cytochrome P450 2A6 (CYP2A6), cytochrome P450 2C8 (CYP2C8), cytochrome P450 2C9 (CYP2C9), cytochrome P450 family 2 subfamily D member 6 (CYP2D6),Cytochrome P450 2E1 (CYP2E1), phylloquinone omega-hydroxylase CYP4F2 (CYP4F2), 7-alpha-hydroxycholest-4-en-3-one 12-alpha-hydroxylase (CYP8B1), dipeptidyl peptidase 4 (DPP4), coagulation factor 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 hydroxyacid oxidase 3 (FMO3), flavin-containing monooxygenase 5 (FMO5), group specific component (vitamin D binding protein) (GC), growth hormone receptor (GHR), glycine N-methyltransferase (GNMT), hyaluronan binding protein 2 (HABP2), hepcidin antimicrobial peptide (HAMP), hydroxyacid oxidase (glycolate oxidase) 1 (HAO1), HGF activator (HGFAC), haptoglobin-related protein; haptoglobin (HPR), hemopexin (HPX), histidine lysate HGF glycoprotein (HRG), hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1), hydroxysteroid (17-beta) dehydrogenase 13 (HSD17B13), inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1), inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), prekallikrein (KLKB1), lactate dehydrogenase A ( LDHA), liver-expressed antimicrobial peptide 2 (LEAP2), leukocyte cell-derived chemotaxin 2 (LECT2), lipoprotein(a) (LPA), mannan-binding lectin serine peptidase 2 (MASP2), S-adenosylmethionine synthase isoform 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 transport 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).

[0183] In addition to their usefulness in silencing the expression of any of the above-mentioned genes for therapeutic purposes, certain nucleic acids (e.g., siRNA) described herein are also useful in research and development applications, as well as diagnostic, preventative, prognostic, clinical, and other health care applications.As a non-limiting example, certain nucleic acids (e.g., siRNA) can be used in target validation tests aimed at testing whether a gene of interest is a potential therapeutic target.Certain nucleic acids (e.g., siRNA) can also be used in target identification tests aimed at finding genes as potential therapeutic targets.

[0184] CRISPR Targeted genome editing has evolved from a niche technology into a method used by many biological researchers. This evolution has been greatly facilitated by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR) technology (see, for example, Sander et al., Nature Biotechnology, 32(4), 347-355 (2014), including supplementary information, International Publication Nos. WO2016 / 197132 and WO2016 / 197133). Therefore, the present specification provides improvements (e.g., lipid nanoparticles and their formulations) that can be used in combination with CRISPR technology to treat diseases such as HBV. Regarding targets for using CRISPR, guide RNAs (gRNAs) used in CRISPR technology can be designed to specifically target identified sequences, such as target genes (e.g., target genes in the HBV genome). Examples of such target sequences are provided in International Publication No. WO2016 / 197132. Furthermore, International Publication No. WO2013 / 151665 (see, e.g., Table 6, which document 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 may also utilize CRISPR technology to target the expression of the target genes discussed herein.

[0185] aiRNA Similar to siRNA, asymmetric interfering RNA (aiRNA) can effectively silence various genes in mammalian cells by recruiting the RNA-induced silencing complex (RISC) and mediating sequence-specific cleavage of 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, aiRNA molecules comprise a short RNA duplex having a sense strand and an antisense strand, with the duplex containing overhangs at the 3' and 5' ends of the antisense strand. aiRNAs are generally asymmetric because the sense strand is shorter at both ends compared to the complementary antisense strand. In some embodiments, aiRNA molecules can be designed, synthesized, and annealed under conditions similar to those used for siRNA molecules. As a non-limiting example, aiRNA sequences can be selected and generated using the methods described above for selecting siRNA sequences.

[0186] 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 usually 12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs) can be designed to target an mRNA of interest, including overhangs at the 3' and 5' ends of the antisense strand. In certain cases, the sense strand of the aiRNA molecule is about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 nucleotides in length, more usually 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 usually about 15-30, 15-25, or 19-25 nucleotides in length, and preferably about 20-24, 21-22, or 21-23 nucleotides in length.

[0187] 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. By way of non-limiting example, the aiRNA sequence may include one or more of the modified nucleotides described above for siRNA sequences. In preferred embodiments, the aiRNA molecule includes 2'OMe nucleotides, such as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, or mixtures thereof.

[0188] In certain embodiments, the aiRNA molecule may comprise an antisense strand corresponding to the antisense strand of an siRNA molecule, such as one of the siRNA molecules described herein. In other embodiments, the aiRNA molecule may be used to silence the expression of any of the target genes described above, such as genes associated with viral infection and survival, genes associated with metabolic diseases and disorders, genes associated with tumorigenesis and cell transformation, angiogenesis genes, immunoregulatory genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative diseases.

[0189] miRNA Generally, microRNAs (miRNAs) are single-stranded RNA molecules approximately 21–23 nucleotides long that regulate gene expression. Although miRNAs are encoded by genes on the DNA from which they are transcribed, 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 then processed into a functional mature miRNA. Mature miRNA molecules are either partially or fully complementary to one or more messenger RNA (mRNA) molecules, and their primary function is to downregulate gene expression. Identification of miRNA molecules 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.

[0190] Genes encoding miRNAs are much longer than the mature miRNA molecules they are processed into. miRNAs are first transcribed as primary transcripts or pri-miRNAs with caps and poly(A) tails, which are then processed in the cell nucleus into short stem-loop structures of approximately 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 through interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC) (Bernstein et al., Nature, 409:363-366 (2001)). Either the sense or antisense strand of DNA can serve as a template for generating miRNAs.

[0191] When Dicer cleaves the pre-miRNA stem loop, two complementary short RNA molecules are formed, but only one of them is incorporated into the RISC complex. This strand, known as the guide strand, is selected by the catalytically active RNase, Argonaute, 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 antiguide 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.

[0192] Mammalian miRNA molecules are typically complementary to sites within the 3'UTR of target mRNA sequences. In certain cases, annealing of miRNA to target mRNA inhibits protein translation by blocking the protein translation machinery. In other specific cases, annealing of miRNA to target mRNA promotes cleavage and degradation of the target mRNA through a process similar to RNA interference (RNAi). miRNAs can also target methylation of genomic sites corresponding to the targeted mRNA. Generally, miRNAs function in association with a complement of proteins collectively referred to as miRNPs.

[0193] 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 usually 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 molecule may comprise one or more modified nucleotides. By way of non-limiting example, the miRNA sequence may include one or more of the modified nucleotides described above for siRNA sequences. In preferred embodiments, the miRNA molecule comprises 2'OMe nucleotides, such as 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, or mixtures thereof.

[0194] 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, immunoregulatory genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.

[0195] In other embodiments, one or more agents that block the activity of miRNA that targets target mRNA of interest are administered using lipid particles (for example, 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 the miRNA binding site on target mRNA.

[0196] antisense oligonucleotides In one embodiment, the nucleic acid is an antisense oligonucleotide directed to a target gene or sequence of interest. The terms "antisense oligonucleotide" or "antisense" include oligonucleotides complementary to a targeted polynucleotide sequence. Antisense oligonucleotides are single strands of DNA or RNA complementary to a selected sequence. Antisense RNA oligonucleotides block translation of a complementary RNA strand by binding to that RNA. Antisense DNA oligonucleotides can be used to target specific complementary (coding or non-coding) RNA. Upon binding, the DNA / RNA hybrid can be degraded by the enzyme RNase H. In certain embodiments, antisense oligonucleotides contain about 10 to about 60 nucleotides, more preferably about 15 to about 30 nucleotides. The term also encompasses antisense oligonucleotides that may not be exactly complementary to the desired target gene. Thus, the present invention can be utilized when non-target specific activity is observed in antisense or when an antisense sequence containing one or more mismatches with the target sequence is most preferred for a particular use.

[0197] Antisense oligonucleotide has been shown to be an effective and targeted inhibitor of protein synthesis, and therefore can be used to specifically inhibit the protein synthesis of targeted genes.The effectiveness of antisense oligonucleotide for inhibiting protein synthesis has been well established.For example, the synthesis of polygalacturonase and muscarinic type 2 acetylcholine receptor is inhibited by the antisense oligonucleotide of their respective mRNA sequence (see United States Patent No. 5,739,119 and United States Patent No. 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.

[0198] Methods for generating antisense oligonucleotides are known in the art and can be readily adapted to generate antisense oligonucleotides targeting any polynucleotide sequence. The selection of an antisense oligonucleotide sequence specific for a given target sequence depends on the analysis of the selected target sequence, as well as the secondary structure, T mThe selection of antisense oligonucleotides is based on the determination of their binding energy, binding energy, and relative stability. Antisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins, or other secondary structures that reduce or prevent specific binding to target mRNA in host cells. Highly preferred target regions of mRNA include the region of or near the AUG translation initiation codon and sequences that are substantially complementary to the 5' region of 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)).

[0199] Ribozymes According to another embodiment of the present invention, the nucleic acid-lipid particle is associated with a ribozyme.Ribozyme is an RNA-protein complex with a specific catalytic domain that has endonuclease activity (see Kim et al., Proc. Natl. Acad. Sci. USA, 84:8788-92 (1987) and Forster et al., Cell, 49:211-20 (1987)).For example, many ribozymes promote phosphoester transfer reactions with 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 ribozyme's internal guide sequence ("IGS") prior to chemical reaction.

[0200] Currently, at least six basic types of naturally occurring enzymatic RNA molecules are known. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans under physiological conditions (and thus can cleave other RNA molecules). Generally, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs via the target-binding portion of the enzymatic nucleic acid, which is held in close proximity to the enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes the target RNA, 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 the target RNA abolishes its ability to direct synthesis of the encoded protein. After an enzymatic nucleic acid binds and cleaves its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

[0201] The enzymatic nucleic acid molecule can 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 RNAse P 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 specific substrate binding sites complementary to one or more DNA or RNA regions of the target gene, and that they contain nucleotide sequences within or surrounding the substrate binding site that confer 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.

[0202] The method of producing ribozyme that targets any polynucleotide sequence is known in the art.Ribozyme can be designed as described in, for example, PCT publication WO93 / 23569 and WO94 / 02595, and synthesized for testing in vitro and / or in vivo as described therein.The disclosures of these PCT publications are incorporated herein by reference in their entirety for all purposes.

[0203] 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 herein 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.

[0204] 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), which can induce immune responses 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 a CpG motif, 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).

[0205] Immunostimulatory nucleic acids are considered sequence-nonspecific if they do not need to specifically bind to and reduce the expression of a target sequence in order to elicit an immune response. Thus, certain immunostimulatory nucleic acids may contain a sequence that corresponds to a region of a naturally occurring gene or mRNA, yet still be considered sequence-nonspecific immunostimulatory nucleic acids.

[0206] In one embodiment, the immunostimulatory nucleic acid or oligonucleotide contains at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be unmethylated or methylated. In another embodiment, the immunostimulatory nucleic acid contains at least one CpG dinucleotide with a methylated cytosine. In one embodiment, the nucleic acid contains a single CpG dinucleotide, and the cytosine in the CpG dinucleotide is methylated. In an alternative embodiment, the nucleic acid contains 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 contains multiple CpG dinucleotides, and at least one of the CpG dinucleotides contains a methylated cytosine. Examples of immunostimulatory oligonucleotides suitable for use in the compositions and methods of the present invention are described in PCT Application No. PCT / US08 / 88676, filed December 31, 2008, PCT Publication Nos. WO02 / 069369 and WO01 / 15726, U.S. Patent No. 6,406,705, and Raney et al., J.Pharm.Exper.Ther., 298:1185-92 (2001), the disclosures of which are each incorporated herein by reference in their entirety for all purposes. In certain embodiments, the oligonucleotides used in the compositions and methods of the present invention have a phosphodiester ("PO") or phosphorothioate ("PS") backbone, and / or at least one methylated cytosine residue within a CpG motif.

[0207] mRNA In certain embodiments, the nucleic acid is one or more mRNA molecules (eg, a cocktail of mRNA molecules).

[0208] mRNA modifications 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.

[0209] 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-taurinomethyl -4-thio-uridine, 5-methyl-uridine, 1-methyl-1-pseudouridine, 4-thio-1-methyl-1-pseudouridine, 2-thio-1-methyl-1-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.

[0210] 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- These include 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.

[0211] 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-isopentenyladenosine, N6 -(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0212] In certain embodiments, the modified nucleoside is 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudouridine. The α-thio-substituted phosphate moiety is provided to confer stability to the RNA polymer via an unnatural phosphorothioate backbone linkage. Phosphorothioate RNA has increased nuclease resistance and therefore a longer half-life in the cellular environment. Phosphorothioate-linked nucleic acids are also expected to reduce innate immune responses through relatively weak binding / activation of cellular innate immune molecules.

[0213] In certain embodiments, it is desirable to degrade modified nucleic acids introduced into cells intracellularly, for example, when precise timing of protein production is desired. Accordingly, the present invention provides modified nucleic acids that contain a degradation domain that can act in a directed manner within a cell.

[0214] 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.

[0215] Optional Components of Modified Nucleic Acids In further embodiments, the modified nucleic acid may contain other optional components that may be beneficial in some embodiments. These optional components include, but are not limited to, untranslated regions, Kozak sequences, intronic 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 contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the translatable region. Nucleic acids containing a Kozak sequence are also provided.

[0216] Additionally, nucleic acids are provided that contain one or more intronic nucleotide sequences that can be excised from the nucleic acid.

[0217] Untranslated Regions (UTRs) The untranslated region (UTR) of a gene is transcribed but not translated. The 5'UTR starts at the transcription initiation site and continues up to, but not including, the initiation codon; whereas, the 3'UTR starts immediately after the termination codon and continues up to the transcription termination signal. There is increasing evidence that UTRs play a regulatory role in the stability and translation of nucleic acid molecules. To increase the stability of the molecule, UTR regulatory functions can be incorporated into the mRNA used in the present invention. Specific functions can also be incorporated to ensure that transcripts are down-regulated when misdirected to undesired organ sites.

[0218] 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 associates with poly(A)-binding protein to form mature circular mRNA species, thereby contributing to the stability and translatability of mRNA within the cell. The cap also assists in the removal of 5'-proximal introns during mRNA splicing.

[0219] Endogenous mRNA molecules can be capped at the 5' end, resulting in a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue of the mRNA molecule and the 5'-terminal transcribed sense nucleotide. This 5'-guanylate cap can then be methylated to yield an N7-methyl-guanylate residue. The ribose sugar of the 5'-end and / or pre-terminal transcribed nucleotide of the mRNA can also optionally be 2'-O-methylated. 5'-cap removal by hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0220] IRES sequence mRNAs containing internal ribosome entry sites (IRES) are also useful in practicing the present invention. An IRES may function as the only ribosome binding site or as one of multiple ribosome binding sites in an mRNA. An mRNA containing two or more functional ribosome binding sites can encode several peptides or polypeptides that are independently translated by the ribosome (a "multicistronic mRNA"). When an mRNA is provided with an IRES, a second translatable region is optionally also provided. Examples of IRES sequences that can be used in accordance with 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).

[0221] PolyA tail During RNA processing, a long chain of adenine nucleotides (poly-A tail) may be added to polynucleotides such as mRNA molecules to increase stability. Immediately after transcription, the 3' end of the transcript may be cleaved, freeing 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.

[0222] Generally, 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).

[0223] In this context, the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer in length than the modified mRNA. The poly-A tail can also be designed as part of the modified nucleic acid to which it belongs. In this context, the poly-A 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 poly-A tail.

[0224] Creation of mRNA molecules 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., Molecular Cloning, A Laboratory Manual (2nd ed. 1989)), as are PCR methods (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds., 1990)). Expression libraries are also well known to those skilled in the art. Additional basic documents disclosing general methods for 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 herein by reference in their entireties for all purposes.

[0225] Encoded Polypeptide The mRNA component of the nucleic acid-lipid particles described herein can be used to express a polypeptide of interest. Certain human diseases are caused by the absence or impairment of a functional protein in a cell type in which the protein is normally present and active. A functional protein may be completely or partially absent, for example, due to transcriptional inactivation of the encoding gene or due to the presence of a mutation in the encoding gene that renders the protein completely or partially nonfunctional. Examples of human diseases caused by complete or partial protein inactivation 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 with 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 impairment or death.

[0226] Gene therapy has been used to treat some diseases caused by the lack or damage of functional protein in the cell type in which the protein normally exists and is active.Gene therapy usually involves the introduction of a vector containing a gene encoding the functional form of the affected protein into the affected person, and the expression of the functional protein to treat the disease.So far, the success of gene therapy has been limited.In addition, a specific embodiment of using LNP to deliver mRNA has been described, for example, in International Publication No. WO2018 / 006052 and International Publication No. WO2015 / 011633.

[0227] Thus, there is a continuing need for improvements in expressing functional forms of proteins in humans suffering from diseases caused by the complete or partial absence of the functional protein, and for example, improvements in nucleic acid (e.g., mRNA) delivery via methods and compositions that can induce fewer immune responses 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 the absence 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.

[0228] Self-amplifying RNA In certain embodiments, the nucleic acid is one or more self-amplifying RNA molecules. Self-amplifying RNA (sa-RNA) is also sometimes referred to as self-replicating RNA, replicable RNA, replicon, or RepRNA. When derived from a positive-strand virus, RepRNA, also known as self-amplifying mRNA, is produced from a viral genome lacking at least one structural gene. 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 encoding a desired antigen of interest. For example, the alphavirus genome is divided into two open reading frames (ORFs), with the first ORF encoding the RNA-dependent RNA polymerase (replicase) protein 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 essential part of the vaccine, facilitating intracellular amplification of RNA after immunization.

[0229] 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, hormone therapy drugs, immunotherapy drugs, radiotherapy drugs, etc.), lipid-lowering agents, 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 may be administered alone in the lipid particles of the present invention, or may be administered in combination (e.g., co-administered) with the lipid particles of the present invention containing nucleic acids such as interfering RNA or mRNA.

[0230] 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.), topoisomerase inhibitors ... kinase 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 Anti-cancer drugs (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.), pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogs thereof, and combinations thereof.

[0231] Examples of conventional hormone therapy agents include, but are not limited to, steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, tamoxifen, and goserelin, as well as other gonadotropin-releasing hormone agonists (GnRH).

[0232] 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-specific antibodies include, but are not limited to, anti-CD20 monoclonal antibodies conjugated to I, etc.

[0233] 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 Radionuclides such as, but not limited to, Bi.

[0234] Further oncology drugs that may be used in accordance with the present invention include alkeran, allopurinol, altretamine, amifostine, anastrozole, araC, arsenic trioxide, bexarotene, biCNU, carmustine, CCNU, celecoxib, cladribine, cyclosporin A, cytosine arabinoside, cytoxan, dexrazoxane, DTIC, estramustine, exemestane, FK506, gemtuzumab-ozogamicin, hydrea, hydroxyurea, idarubicin, interferon, letrozole, leustatin, These include, but are not limited to, leuprolide, litretinoin, megastrol, L-PAM, mesna, methoxsalen, mithramycin, nitrogen mustard, pamidronate, pegademase, pentostatin, porfimer sodium, prednisone, rituximab, streptozocin, STI-571, taxotere, temozolamide, VM-26, toremifene, tretinoin, ATRA, valrubicin, and velban. Other examples of oncology drugs that can be used in accordance with the present invention are ellipticine and ellipticine analogs or derivatives, epothilones, intracellular kinase inhibitors, and camptothecin.

[0235] 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.

[0236] 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-λ molecules such as IFN-λ1, IFN-λ2, and IFN-λ3), type II interferons (e.g., IFN-γ), type I interferons (e.g., IFN-α such as PEGylated IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ), interferons, lamibcin, lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogs, 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.

[0237] lipid particles Lipid particles of the present invention typically comprise an active agent or therapeutic agent, an ionizable lipid of the present invention, a non-cationic lipid, and a conjugated lipid that inhibits particle aggregation. In some 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. In other embodiments, the lipid particles described herein are substantially non-toxic to mammals, such as humans. Lipid particles of the present invention typically have an average diameter of about 40 nm to about 150 nm, about 40 nm to about 80 nm, about 40 nm to about 60 nm, about 50 nm to about 80 nm, about 50 nm to about 60 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm.

[0238] In a preferred embodiment, the lipid particles of the present invention are serum-stable nucleic acid-lipid particles (LNPs) comprising one or more nucleic acid molecules, such as interfering RNA (e.g., siRNA, aiRNA, and / or miRNA) or mRNA; an ionizable lipid of the present invention (e.g., a cationic lipid of Formula I); a non-cationic lipid (e.g., cholesterol alone or a mixture of cholesterol with one or more phospholipids); and a conjugated lipid that inhibits particle aggregation (e.g., one or more PEG-lipid conjugates). The LNPs may comprise 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, and PCT Publication No. WO 96 / 40964, the disclosures of each of which are incorporated herein by reference in their entirety for all purposes.

[0239] Ionizable lipids In one embodiment, the present invention provides a compound of formula -NR 2 R 3 an ionizable group of formula (wherein R2 is C1-C4 alkyl, and R 3 is C1-C4 alkyl); 15-40 carbon atoms, 30-80 hydrogen atoms; 0-10 oxygen atoms; 0-10 nitrogen atoms; 0-4 fluorine; and 0-5 sulfur atoms, wherein the ionizable lipid has a clearance rate such that less than 10% of the ionizable parent lipid remains 7 days after administration (e.g., in the assay described in Example 32 herein).

[0240] In one embodiment, the ionizable lipid comprises at least one group of the formula -C(=O)-O-.

[0241] In one embodiment, the ionizable lipid comprises at least two groups of the formula -C(=O)-O-.

[0242] In one embodiment, the ionizable lipid has the formula —(C═O)N(H)N(R 4 )(R 5 )-, wherein R 4 is C2~C 30 is hydrocarbyl; and R 5 is C2~C 30 It is a hydrocarbyl.

[0243] In one embodiment, the ionizable lipid has the formula —(C═O)N(H)—N═C(R 4 )-, wherein R 4 is C2~C 30 is hydrocarbyl; and R 5 is C2~C 30 It is a hydrocarbyl.

[0244] In one embodiment, the ionizable lipid has the formula —(C═O)N(H)N(H)(R 4 )(R 5 )-, wherein R 4 is C2~C 30 is hydrocarbyl; and R 5 is C2~C 30 It is a hydrocarbyl.

[0245] In one embodiment, the ionizable lipid comprises at least one group of formula -CHF-C(=O)-O- or -CF2-C(=O)-O-.

[0246] In one embodiment, X is —CH 2 —.

[0247] In one embodiment, X is -C(=O)-.

[0248] In one embodiment, X is —O—.

[0249] In one embodiment, R 2 is methyl.

[0250] In one embodiment, R 2 is ethyl.

[0251] In one embodiment, R 2 is propyl.

[0252] In one embodiment, R 3 is methyl.

[0253] In one embodiment, R 3 is ethyl.

[0254] In one embodiment, R 3 is propyl.

[0255] In one embodiment, m is 1.

[0256] In one embodiment, m is 2.

[0257] In one embodiment, m is 3.

[0258] In one embodiment, the group: [ka] is selected from the group consisting of (CH)NCHCHC(=O)-, (CH)NCHCHCHC(=O)-, (CH)NCHCHCHCHC(=O)-, (CH)NCHCHCH-, (CH)NCHCHCHCH-, 5-pyrrolidin-1-ylpentanoyl, and (CH)NCHCHCHCHO-.

[0259] In one embodiment, the group: [ka] is selected from the group consisting of (CH3)2NCH2CH2C(=O)-, (CH3)2NCH2CH2CH2C(=O)-, (CH3)2NCH2CH2CH2CH2C(=O)-, (CH3)2NCH2CH2-, (CH3)2NCH2CH2CH-, and (CH3)2NCH2CH2CH2O-.

[0260] In one embodiment, R 1 is C5~C 25 It is a hydrocarbyl.

[0261] In one embodiment, R 1 is C2~C 25 It is alkyl.

[0262] In one embodiment, R 1 is C5~C 25 ) alkyl.

[0263] In one embodiment, R 1 is C5~C 25 It is alkenyl.

[0264] In one embodiment, R 1 is 1-nonyl or 1-decyl.

[0265] In one embodiment, o is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0266] In one embodiment, o is 10, 11, 12, 13, 14, or 15.

[0267] In one embodiment, o is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0268] In one embodiment, o is 2, 3, 4, or 5.

[0269] In one embodiment, L 2 -(C=O)-O-, -(C=O)N(H)N( R4 )-, -(C=O)N(H)-N=C(R 4 )-, -(C=O)N(H)N(H)C(R 4 )(R 5 )-, or -(C=O)-O-CH2-R z is.

[0270] In one embodiment, L 2 is -(C=O)-O-.

[0271] In one embodiment, L 2 is -(C=O)N(H)N(R 4 )-.

[0272] In one embodiment, L 2 is -(C=O)-O-CH2-R z is.

[0273] In one embodiment, a is H.

[0274] In one embodiment, a is F.

[0275] In one embodiment, b is H.

[0276] In one embodiment, b is F.

[0277] In one embodiment, R 6 is C2~C 25 It is a hydrocarbyl.

[0278] In one embodiment, R 6 is C5~C 25 It is a hydrocarbyl.

[0279] In one embodiment, R 6 is C2~C 25 It is alkyl.

[0280] In one embodiment, R 6 is C5~C 25 It is alkyl.

[0281] In one embodiment, R 6 is C5~C 25 It is alkenyl.

[0282] In one embodiment, the group: [ka] teeth, [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0283] In one embodiment, the group: [ka] teeth, [ka] is selected from the group consisting of:

[0284] In one embodiment, Y is —CH 2 —.

[0285] In one embodiment, Y is -C(=O)-.

[0286] In one embodiment, Y is —O—.

[0287] In one embodiment, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0288] In one embodiment, n is 10, 11, 12, 13, 14, or 15.

[0289] In one embodiment, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0290] In one embodiment, n is 2, 3, 4, or 5.

[0291] In one embodiment, L 1 does not exist; and L 2 -(C=O)-O-, -(C=O)N(H)N(R 4 )-, -(C=O)N(H)-N=C(R 4 )-, -(C=O)N(H)N(H)C(R 4 )(R 5 )-, or -(C=O)-O-CH2-R z is.

[0292] In one embodiment, L 1 -(C=O)-O-, -(C=O)N(H)N(R 4 )-, -(C=O)N(H)-N=C(R 4 )-, -(C=O)N(H)N(H)C(R 4 )(R 5 )-, or -(C=O)-O-CH2-R a and L 2 does not exist.

[0293] In one embodiment, L 1 -(C=O)-O-, -(C=O)N(H)N(R 4 )-, -(C=O)N(H)-N=C(R 4 )-, -(C=O)N(H)N(H)C(R 4 )(R5 )-, or -(C=O)-O-CH2-R a is.

[0294] In one embodiment, L 1 is -(C=O)-O.

[0295] In one embodiment, L 1 is -(C=O)N(H)N(R 4 )-.

[0296] In one embodiment, L 1 is -(C=O)-O-CH2-R z is.

[0297] In one embodiment, c is H.

[0298] In one embodiment, c is F.

[0299] In one embodiment, d is H.

[0300] In one embodiment, d is F.

[0301] In one embodiment, R 7 is C2-C 25 It is a hydrocarbyl.

[0302] In one embodiment, R 7 is C5~C 25 It is a hydrocarbyl.

[0303] In one embodiment, R 7 is C2~C 25 It is alkyl.

[0304] In one embodiment, R 7 is C5~C 25 It is alkyl.

[0305] In one embodiment, R 7 is C5~C 25 It is alkenyl.

[0306] In one embodiment, the group: [ka] teeth, [ka] [ka] is selected from the group consisting of:

[0307] In one embodiment, the group: [ka] teeth, [ka] is selected from the group consisting of:

[0308] In one embodiment, R 4 is C2~C 25 It is a hydrocarbyl.

[0309] In one embodiment, R 4 is C5~C 25 It is a hydrocarbyl.

[0310] In one embodiment, R 4 is C2~C 25 It is alkyl.

[0311] In one embodiment, R 4 is C5~C 25 It is alkyl.

[0312] In one embodiment, R 4 is C5~C 25 It is alkenyl.

[0313] In one embodiment, R 5 is C2~C 25 It is a hydrocarbyl.

[0314] In one embodiment, R 5 is C5~C 25 It is a hydrocarbyl.

[0315] In one embodiment, R 5 is C2~C 25 It is alkyl.

[0316] In one embodiment, R 5 is C5~C 25 It is alkyl.

[0317] In one embodiment, R 5 is C5~C 25 It is alkenyl.

[0318] In one embodiment, R 4’ is C2-C 25 It is a hydrocarbyl.

[0319] In one embodiment, R' 4 is C5-C 25 It is a hydrocarbyl.

[0320] In one embodiment, R 4’ is C2~C 25 It is alkyl.

[0321] In one embodiment, R 4’ is C5~C 25 It is alkyl.

[0322] In one embodiment, R 4’ is C5~C 25 It is alkenyl.

[0323] In one embodiment, R' 5 is C2~C 25 It is a hydrocarbyl.

[0324] In one embodiment, R' 5’ is C5~C 25 It is a hydrocarbyl.

[0325] In one embodiment, R 5’is C2~C 25 It is alkyl.

[0326] In one embodiment, R 5 is C5~C 25 It is alkyl.

[0327] In one embodiment, R 5’ is C5~C 25 It is alkenyl.

[0328] In one embodiment, R z is C2~C 10 In one embodiment, each C2 to C6 is phenyl substituted with 1 or 2 groups independently selected from hydrocarbyl. 10 Hydrocarbyl is C5 to C 10 In one embodiment, each C to C is independently selected from the group consisting of: 10 Hydrocarbyl is C2 to C 10 In one embodiment, each C2 to C6 is independently selected from the group consisting of alkyl. 10 Hydrocarbyl is C5 to C 10 In one embodiment, each C2 to C6 is independently selected from the group consisting of alkyl, 10 Hydrocarbyl is C5 to C 10 alkenyl.

[0329] In one embodiment, R z’ is C2~C 10 In one embodiment, each C2 to C6 is phenyl substituted with 1 or 2 groups independently selected from hydrocarbyl. 10 Hydrocarbyl is C5 to C 10 In one embodiment, each C to C is independently selected from the group consisting of: 10 Hydrocarbyl is C2 to C 10 In one embodiment, each C2 to C6 is independently selected from the group consisting of alkyl, 10 Hydrocarbyl is C5 to C 10 In one embodiment, each C2 to C6 is independently selected from the group consisting of alkyl, 10 Hydrocarbyl is C5 to C 10alkenyl.

[0330] In one embodiment, the ionizable lipid is selected from the compounds of Examples 1, 16, 17, 43, 46, 90, 101, and 115, and salts thereof.

[0331] In one embodiment, X is -C(=O)- and Y is absent or -O-.

[0332] In one embodiment, X is -C(=O)- and Y is -O-.

[0333] In one embodiment, Y is —CH— and L 1 does not exist, and c and d are each H.

[0334] In one embodiment, a is F or b is H.

[0335] In one embodiment, a is F and b is F.

[0336] In one embodiment, a is F; b is H; and L 1 is -(C=O)-O-.

[0337] In one embodiment, a is F; b is F; and L 1 is -(C=O)-O-.

[0338] In one embodiment, R 2 and R 3 are each methyl.

[0339] In one embodiment, L 1 does not exist; c and d are each H.

[0340] In one embodiment, L 1 does not exist; a, b, c and d are each H.

[0341] In one embodiment, L 1and L 2 One of the groups is —(C═O)—O—.

[0342] In one embodiment, L 1 is -(C=O)-O-, and R 7 is branched chain C2~C 25 It is a hydrocarbyl.

[0343] In one embodiment, L 1 is -(C=O)-O-, and R 7 L 1 C5-C branched within 3 carbons of the bond point to 25 It is a hydrocarbyl.

[0344] In one embodiment, L 1 is -(C=O)-O-, and R 7 L 1 C4-C branched within 2 carbons of the bond point to 25 It is a hydrocarbyl.

[0345] In one embodiment, L 1 is -(C=O)-O-, and R 7 L 1 C3 to C branched at the carbon bonded to 25 It is a hydrocarbyl.

[0346] In one embodiment, L 1 and L 2 is -(C=O)-O-, and R 7 is branched chain C2~C 25 It is a hydrocarbyl.

[0347] In one embodiment, L 1 and L 2 is -(C=O)-O-, and R 7 L 1 C5-C branched within 3 carbons of the bond point to 25 It is a hydrocarbyl.

[0348] In one embodiment, L 1 and L 2is -(C=O)-O-, and R 7 L 1 C4-C branched within 2 carbons of the bond point to 25 It is a hydrocarbyl.

[0349] In one embodiment, L 1 and L 2 is -(C=O)-O-, and R 7 L 1 C3 to C branched at the carbon bonded to 25 It is a hydrocarbyl.

[0350] In one embodiment, Y is absent or is —O—; N is 0; c is H; d is H; and L 1 does not exist;R 7 is C2~C 25 It is a hydrocarbyl.

[0351] In one embodiment, Y is absent or is —O—; N is 0; c is H; d is H; and L 1 does not exist;R 7 is C5~C 15 It is a hydrocarbyl.

[0352] In one embodiment, Y is absent or is —O—; N is 0; c is H; d is H; and L 1 does not exist;R 7 is C 10 It is a hydrocarbyl.

[0353] In one embodiment, Y is absent or is -O-; N is 0; c is H; d is H; and L 1 does not exist;R 7 is a C5-C branched at a carbon atom within 3 carbon atoms of the point of attachment to the remainder of the compound of formula (I); 15 It is a hydrocarbyl.

[0354] In one embodiment, Y is absent or is —O—; N is 0; c is H; d is H; and L 1 does not exist;R7 is a C5-C branched at a carbon atom within two carbon atoms of the point of attachment to the remainder of the compound of formula (I); 15 It is a hydrocarbyl.

[0355] In one embodiment, Y is absent or is —O—; N is 0; c is H; d is H; and L 1 does not exist;R 7 is a C5-C branched at the carbon attached to the remainder of the compound of formula (I) 15 It is a hydrocarbyl.

[0356] In one embodiment, Y is absent or is -O-; N is 0; c is H; d is H; and L 1 does not exist;R 7 is a C branched at a carbon within 3 carbons of the point of attachment to the remainder of the compound of formula (I); 10 It is a hydrocarbyl.

[0357] In one embodiment, Y is absent or is -O-; N is 0; c is H; d is H; and L 1 does not exist;R 7 is a C branched at a carbon within two carbons of the point of attachment to the remainder of the compound of formula (I); 10 It is a hydrocarbyl.

[0358] In one embodiment, Y is absent or is —O—; N is 0; c is H; d is H; and L 1 does not exist;R 7 is a C branched at the carbon attached to the remainder of the compound of formula (I) 10 It is a hydrocarbyl.

[0359] In one embodiment, L 2 is -(C=O)-O-, and R 6 is branched chain C2~C 25 It is a hydrocarbyl.

[0360] In one embodiment, L 2 is -(C=O)-O-, and R 6 L2 C5-C branched within 3 carbons of the bond point to 25 It is a hydrocarbyl.

[0361] In one embodiment, L 2 is -(C=O)-O-, and R 6 L 2 C4-C branched within 2 carbons of the bond point to 25 It is a hydrocarbyl.

[0362] In one embodiment, L 2 is -(C=O)-O-, and R 6 L 2 C3 to C branched at the carbon bonded to 25 It is a hydrocarbyl.

[0363] In one embodiment, L 2 is -(C=O)-O-; a is F; b is H; R 6 is branched chain C2~C 25 It is a hydrocarbyl.

[0364] In one embodiment, L 2 is -(C=O)-O-; a is F; b is H; R 6 L 2 C5-C branched within 3 carbons of the bond point to 25 It is a hydrocarbyl.

[0365] In one embodiment, L 2 is -(C=O)-O-; a is F; b is H; R 6 L 2 C4-C branched within 2 carbons of the bond point to 25 It is a hydrocarbyl.

[0366] In one embodiment, L 2 is -(C=O)-O-; a is F; b is H; R 6 L 2 C3 to C branched at the carbon bonded to 25 It is a hydrocarbyl.

[0367] In one embodiment, L 2 is -(C=O)-O-; a is F; b is F; R 6 is branched chain C2~C 25 It is a hydrocarbyl.

[0368] In one embodiment, L 2 is -(C=O)-O-; a is F; b is F; R 6 L 2 C5-C branched within 3 carbons of the bond point to 25 It is a hydrocarbyl.

[0369] In one embodiment, L 2 is -(C=O)-O-; a is F; b is F; R 6 L 2 C4-C branched within 2 carbons of the bond point to 25 It is a hydrocarbyl.

[0370] In one embodiment, L 2 is -(C=O)-O-; a is F; b is F; R 6 L 2 C3 to C branched at the carbon bonded to 25 It is a hydrocarbyl.

[0371] In one embodiment, the present invention provides a compound of formula (I): [ka] or a salt thereof, wherein: X is -CH2-, -O-, -O(C=O), or -C(=O)-; Y is -CH2-, -O-, -SO2-, or -C(=O)-; R 1 is C2~C 25 is a hydrocarbyl; R 2 is C1-C3 alkyl, and R 3 is C1-C3 alkyl; or R 2 and R3 together with the nitrogen to which they are attached form an aziridino, azetidino, or pyrrolidino ring; m is 0, 1, 2, 3, 4, 5, or 6; n is 0 to 15; o is 0 to 15; L 1 does not exist, -(C=O)-O-, -(C=O)N(H)N(R 4 )-, -(C=O)N(H)-N=C(R 4 )-, -(C=O)N(H)N(H)C(R 4 )(R 5 )-, -(C=O)-O-CH2-R z , or -O(C=O)-R w - and; L 2 does not exist, -(C=O)-O-, -(C=O)N(H)N(R 4’ )-, -(C=O)N(H)-N=C(R 4’ )-, -(C=O)N(H)N(H)C(R 4’ )(R 5’ )-, -(C=O)-O-CH2-R z’ , or -O(C=O)-R x - and; a is H or F; b is H or F; c is H or F and d is H or F, or c and d together with the carbon to which they are attached form -C(=O)-; R 4 is H or C2~C 25 is a hydrocarbyl; R 5 is H or C2~C 25 is a hydrocarbyl; R 4’ is H or C2~C 25 is a hydrocarbyl; R 5’ is H or C2~C 25 is a hydrocarbyl; R 6 is C2~C 25 is a hydrocarbyl; R7 is C2~C 25 is a hydrocarbyl; R w is absent, -C(F)H-, or -C(F)2-; R x is absent, -C(F)H-, or -C(F)2-; R z is C2~C 10 phenyl substituted with 1, 2, or 3 groups independently selected from hydrocarbyl; R z’ is C2~C 10 and phenyl substituted with 1, 2, or 3 groups independently selected from hydrocarbyl.

[0372] In one embodiment, the present invention provides a compound of formula (I): [ka] or a salt thereof, wherein: X is -CH2-, -O-, -O(C=O), or -C(=O)-; Y is -CH2-, -O-, -SO2-, or -C(=O)-; R 1 is C2~C 25 is a hydrocarbyl; R 2 is C1-C3 alkyl, and R 3 is C1-C3 alkyl; or R 2 and R 3 together with the nitrogen to which they are attached form an aziridino, azetidino, or pyrrolidino ring; m is 0, 1, 2, 3, 4, 5, or 6; n is 0 to 15; o is 0 to 15; L 1 is absent or is -(C=O)-O-; L 2 is absent or is -(C=O)-O-; a is H or F; b is H or F; c is H or F and d is H or F, or c and d together with the carbon to which they are attached form -C(=O)-; R 4 is H or C2~C 25 is a hydrocarbyl; R 5 is H or C2~C 25 is a hydrocarbyl; R 4’ is H or C2~C 25 is a hydrocarbyl; R 5’ is H or C2~C 25 is a hydrocarbyl; R 6 is C2~C 25 is a hydrocarbyl; R 7 is C2-C 25 is a hydrocarbyl; R w is absent, -C(F)H-, or -C(F)2-; R x is absent, -C(F)H-, or -C(F)2-; R z is C2-C 10 phenyl substituted with 1, 2, or 3 groups independently selected from hydrocarbyl; and R z’ is C2-C 10 and phenyl substituted with 1, 2, or 3 groups independently selected from hydrocarbyl.

[0373] Specific Embodiments In one particular embodiment, the present invention provides ionizable lipids of formula (I) where X is -C(=O)-. Such ionizable lipids typically exhibit high activity.

[0374] In a specific embodiment, the present invention provides an ionizable lipid of formula (I) wherein X is -C(=O)- and Y is -O-. Such ionizable lipids typically exhibit high activity and have desirable pKa properties.

[0375] In one specific embodiment, the present invention provides an ionizable lipid of formula (I), wherein Y is —CH—; L 1 is absent, and c and d are each H. Such ionizable lipids usually exhibit high activity.

[0376] In a specific embodiment, the present invention provides an ionizable lipid of formula (I), wherein R 2 is methyl and R 3 is methyl. Such ionizable lipids usually exhibit high activity.

[0377] In a specific embodiment, the present invention provides an ionizable lipid of formula (I), wherein L 1 is absent, and c and d are each H. Such ionizable lipids usually exhibit high activity.

[0378] In a specific embodiment, the present invention provides an ionizable lipid of formula (I), wherein L 2 is -(C=O)-O-, and R 6 L 2 branched at the carbon atom attached to L 2 Branched at a carbon atom that is 2 carbons from the point of attachment to L, or 2 C2-C branched at the carbon atom that is 3 carbons from the point of attachment to 25 Such ionizable lipids typically exhibit high activity.

[0379] Hydrocarbyl is L 1 Or L 2 branched at the carbon atom attached to L 1 Or L 2 Branched at a carbon atom that is 2 carbons from the point of attachment to L, or 1 Or L 2 When branched at a carbon atom that is 3 carbons from the point of attachment to, the hydrocarbyl is understood to be branched at one of the carbons shown in the formula: [ka]

[0380] In one embodiment, the present invention provides (a) one or more nucleic acid molecules; (b) non-cationic lipids; (c) complex lipids; (d) a compound of formula (I) or a salt thereof, wherein the one or more nucleic acid molecules are encapsulated within the lipid particle.

[0381] In one embodiment, the compound or salt comprises from 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; and the complex lipid comprises from about 0.1 mol% to about 10 mol% of the total lipid present in the particle.

[0382] In one embodiment, the compound or salt comprises from 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; and the complex lipid comprises from about 0.1 mol% to about 10 mol% of the total lipid present in the particle.

[0383] In one embodiment, 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.

[0384] In one embodiment, the nucleic acid is an mRNA molecule.

[0385] In one embodiment, the nucleic acid comprises a double-stranded siRNA molecule.

[0386] In one embodiment, the double-stranded siRNA molecule comprises at least one modified nucleotide.

[0387] In one embodiment, the siRNA comprises at least one 2'-O-methyl (2'OMe) nucleotide.

[0388] In one embodiment, the non-cationic lipid comprises cholesterol or a derivative thereof.

[0389] In one embodiment, cholesterol or a derivative thereof comprises about 31.5 mol % to about 42.5 mol % of the total lipid present in the particle.

[0390] In one embodiment, the non-cationic lipid comprises a phospholipid.

[0391] In one embodiment, the non-cationic lipid comprises a mixture of a phospholipid and cholesterol or a derivative thereof.

[0392] In one embodiment, the phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or a mixture thereof.

[0393] In one embodiment, the phospholipids constitute about 4 mol % to about 10 mol % of the total lipids present in the particles, and the cholesterol constitutes about 30 mol % to about 40 mol % of the total lipids present in the particles.

[0394] In one embodiment, the phospholipids constitute about 10 mol % to about 30 mol % of the total lipids present in the particles, and the cholesterol constitutes about 10 mol % to about 30 mol % of the total lipids present in the particles.

[0395] In one embodiment, the conjugated lipid comprises a polyethylene glycol (PEG)-lipid conjugate.

[0396] In one embodiment, the PEG-lipid conjugate has formula (I): [ka] or a salt thereof, wherein R 1is H, (C1-C6) alkyl, or (C1-C6) alkanoyl; n is an integer ranging from about 10 to about 150; L is absent, X is -C(=O)NR 2 R 3 or L is (C1-C6) alkyl and X is -N(R 4 )C(=O)CH(R 2 )(R 3 ), -OCH(R 2 )(R 3 ), -C(=O)OCH2CH(R 2 )(R 3 ), -N(R 4 )C(=O)N(R 2 )(R 3 ), and -SO2N(R 2 )(R 3 ) selected from the group consisting of; R 2 (C 10 -C 20 ) alkyl; R 3 (C 10 -C 20 ) alkyl; and R 4 is H or (C1-C6) alkyl.

[0397] In one embodiment, the PEG-lipid conjugate is [ka] The present invention includes a compound selected from the group consisting of:

[0398] In one embodiment, the PEG-lipid conjugate has the following formula: (I): [ka] In the formula, n is 45 to 50.

[0399] In one embodiment, the PEG-lipid conjugate comprises a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, or a mixture thereof.

[0400] In one embodiment, the PEG-DAA conjugate comprises a PEG-dimyristyloxypropyl (PEG-DMA) conjugate, a PEG-distearyloxypropyl (PEG-DSA) conjugate, or a mixture thereof.

[0401] In one embodiment, the PEG-DAA conjugate comprises a PEG-dimyristyloxypropyl (PEG-DMA) conjugate.

[0402] In one embodiment, the polyethylene glycol (PEG)-lipid conjugate is a compound of the following formula: ABC or a salt thereof, wherein 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 (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. alkoxy is substituted with one or more anion precursor groups, and wherein any of the (C-C)alkyl, (C-C)cycloalkyl, (C-C)cycloalkyl(C-C)alkyl, (C-C)alkoxy, (C-C)alkenyl, (C-C)alkynyl, (C-C)alkanoyl, (C-C)alkoxycarbonyl, (C-C)alkylthio, and (C-C)alkanoyloxy is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, (C-C)alkoxy, (C-C)alkanoyl, (C-C)alkoxycarbonyl, (C-C)alkylthio, or (C-C)alkanoyloxy; B is a polyethylene glycol chain having a molecular weight of about 550 daltons to about 10,000 daltons; C is -LR a and L is a direct bond, -C(O)O-, or -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)CCHCHC(O)-, and -NHC(O)CHCHC(O)NH-; R a is a branched (C 10 -C50 ) alkyl or branched (C 10 -C 50 ) alkenyl, wherein said branched (C 10 -C 50 ) alkyl or branched (C 10 -C 50 ) one or more carbon atoms of the alkenyl are replaced with -O-; R b are each independently H or (C1-C6) alkyl.

[0403] In one embodiment, the PEG has an average molecular weight of about 2,000 daltons.

[0404] In one embodiment, the complex lipids comprise about 1 mol % to about 2 mol % of the total lipids present in the particle.

[0405] In one embodiment, the nucleic acid in the nucleic acid-lipid particles is not substantially degraded after incubating the particles in serum at 37° C. for 30 minutes.

[0406] In one embodiment, the nucleic acid is fully encapsulated in the nucleic acid-lipid particle.

[0407] In one embodiment, the nucleic acid-lipid particles have a lipid:nucleic acid mass ratio of about 5 to about 15.

[0408] In one embodiment, the nucleic acid-lipid particles have a lipid:nucleic acid mass ratio of about 5 to about 30.

[0409] In one embodiment, the nucleic acid-lipid particles have a median diameter of about 40 nm to about 150 nm.

[0410] In one embodiment, the compound or salt comprises 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 comprises about 31.5 mol% to about 42.5 mol% of the total lipid present in the particle; and the PEG-lipid conjugate comprises about 1 mol% to about 2 mol% of the total lipid present in the particle.

[0411] In one embodiment, the nucleic acid-lipid particles comprise about 61.5 mol % of the compound or salt, about 36.9% of cholesterol or a derivative thereof, and about 1.5 mol % of the PEG-lipid conjugate.

[0412] In one embodiment, the compound or salt constitutes about 52 mol% to about 62 mol% of the total lipid present in the particle; the non-cationic lipid comprises a mixture of phospholipid and cholesterol or a derivative thereof, which constitutes about 36 mol% to about 47 mol% of the total lipid present in the particle; and the PEG-lipid conjugate constitutes about 1 mol% to about 2 mol% of the total lipid present in the particle.

[0413] In one embodiment, the nucleic acid-lipid particles comprise about 57.1 mol % of the compound or salt, about 7.1 mol % of a phospholipid, about 34.3 mol % of cholesterol or a derivative thereof, and about 1.4 mol % of a PEG-lipid conjugate.

[0414] In one embodiment, the nucleic acid-lipid particles comprise about 57.1 mol % of the compound or salt, about 20 mol % of a phospholipid, about 20 mol % of cholesterol or a derivative thereof, and about 1.4 mol % of a PEG-lipid conjugate.

[0415] In one embodiment, the compound or salt constitutes 50 mol% to 65 mol% of the total lipids present in the particle; the non-cationic lipid comprises a mixture of phospholipids and cholesterol or a derivative thereof, the phospholipids constituting 4 mol% to 10 mol% of the total lipids present in the particle, and the cholesterol or a derivative thereof constituting 30 mol% to 40 mol% of the total lipids present in the particle; and the complex lipids constituting 0.5 mol% to 2 mol% of the total lipids present in the particle.

[0416] In one embodiment, the compound or salt constitutes 50 mol% to 65 mol% of the total lipids present in the particle; the non-cationic lipid comprises a mixture of phospholipids and cholesterol or a derivative thereof, the phospholipids constituting 3 mol% to 15 mol% of the total lipids present in the particle, and the cholesterol or a derivative thereof constituting 30 mol% to 40 mol% of the total lipids present in the particle; and the complex lipids constituting 0.5 mol% to 2 mol% of the total lipids present in the particle.

[0417] In one embodiment, the compound or salt constitutes 50 mol% to 65 mol% of the total lipid present in the particle; the non-cationic lipid constitutes up to 49.5 mol% of the total lipid present in the particle; the cholesterol or derivative thereof constitutes 30 mol% to 40 mol% of the total lipid present in the particle; and the complex lipid constitutes 0.5 mol% to 2 mol% of the total lipid present in the particle.

[0418] In one embodiment, the compound or salt 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; and the complex lipid constitutes 0.5 mol% to 2 mol% of the total lipid present in the particle.

[0419] In one embodiment, the compound or salt constitutes about 30 mol% to about 50 mol% of the total lipid present in the particle; the non-cationic lipid comprises a mixture of phospholipid and cholesterol or a derivative thereof, which constitutes about 47 mol% to about 69 mol% of the total lipid present in the particle; and the complex lipid constitutes about 1 mol% to about 3 mol% of the total lipid present in the particle.

[0420] In one embodiment, the non-cationic lipid comprises a mixture of cholesterol and DSPC, and the complex lipid comprises [ka] where n is selected so that the resulting polymer chain has a molecular weight of about 2000 daltons, the complex lipid constitutes about 2 mol% of the total lipid present in the particle; DSPC constitutes about 10 mol% of the total lipid present in the particle; cholesterol constitutes about 48 mol% of the total lipid present in the particle; and the compound or salt constitutes about 40 mol% of the total lipid present in the particle.

[0421] In one embodiment, the non-cationic lipid comprises a mixture of cholesterol and DSPC; [ka] where n is selected so that the resulting polymer chain has a molecular weight of about 2000 daltons, the complex 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; and the compound or salt constitutes about 40 mol% of the total lipid present in the particle.

[0422] In one embodiment, the non-cationic lipid comprises a mixture of cholesterol and DSPC, and the complex lipid comprises [ka] where n is selected so that the resulting polymer chain has a molecular weight of about 2000 daltons; the complex lipid constitutes 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; and the compound or salt constitutes about 54.9 mol% of the total lipid present in the particle.

[0423] In one embodiment, the lipid to nucleic acid ratio is about 24.

[0424] In one embodiment, the nucleic acid-lipid particle comprises two or more different compounds of Formula (I) or salts thereof.

[0425] 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.

[0426] Non-limiting examples of non-cationic lipids include 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-phosphatidylethanolamine (DPPG ... Phospholipids include phospholipids such as palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine (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 group in these lipids is preferably C 10 -C 24Acyl groups derived from fatty acids having carbon chains, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0427] 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.

[0428] In some embodiments, the non-cationic lipid present in the lipid particle (e.g., LNP) comprises or consists of cholesterol or a derivative thereof, e.g., a phospholipid-free lipid particle formulation. In other embodiments, the non-cationic lipid present in the lipid particle (e.g., LNP) comprises or consists of one or more phospholipids, e.g., a cholesterol-free lipid particle formulation. In further embodiments, the non-cationic lipid present in the lipid particle (e.g., LNP) comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof.

[0429] Other examples of non-cationic lipids suitable for use in the present invention include phosphorus-free lipids such as, for example, linoleyl alcohol, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic acid polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, ceramides, sphingomyelin, and the like.

[0430] In some embodiments, the non-cationic lipid comprises 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.

[0431] 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.

[0432] In certain other embodiments, the cholesterol present in lipid particles 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 cholesterol at about 34 mol% of the total lipid present in the particle. As other non-limiting examples, the particle may contain cholesterol at 33% or 38.5%.

[0433] 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 comprise cholesterol at about 20 mol% of the total lipid present in the particle.

[0434] 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 from about 2 mol% to about 20 mol%, from about 2 mol% to about 15 mol%, from about 2 mol% to about 12 mol%, from about 2 mol% to about 10 mol%, from about 2 mol% to about 9 mol%, from about 2 mol% to about 8 mol%, from 4 mol% to about 20 mol%, from about 4 mol% to about 15 mol%, from about 4 mol% to about 12 mol%, from about 4 mol% to about 10 mol%, or from about 4 mol% to about 60 mol% of the total lipid present in the particle. It may comprise about 9 mol%, about 4 mol% to about 8 mol%, about 6 mol% to about 20 mol%, about 6 mol% to about 15 mol%, about 6 mol% to about 12 mol%, about 6 mol% to about 10 mol%, about 6 mol% to about 9 mol%, about 2 mol% to about 8 mol%, 8 mol% to about 20 mol%, about 8 mol% to about 15 mol%, about 8 mol% to about 12 mol%, about 8 mol% to about 10 mol%, or about 8 mol% to about 9 mol%. As a non-limiting example, a lipid particle comprising a mixture of phospholipids and cholesterol may comprise a phospholipid such as DPPC or DSPC at about 7 mol% 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 10 mol% to about 15 mol%, about 15 mol% to about 20 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 a phospholipid such as DPPC or DSPC at about 20 mol% of the total lipid present in the particle (e.g., in a mixture with about 20 mol% cholesterol).

[0435] lipid complex In addition to cationic lipid and non-cationic lipid, lipid particles (for example, LNP) of the present invention comprise lipid complexes.The lipid complexes are useful in that they prevent particle aggregation.Suitable lipid complexes include, but are not limited to, PEG-lipid complexes, ATTA-lipid complexes, cationic polymer-lipid complexes (CPL), POZ polyoxazoline (POZ)-lipids, polysarcosine (pSAR)-lipids, and their mixtures.In certain embodiments, particles comprise either PEG-lipid complexes or ATTA-lipid complexes together with CPL.

[0436] In a preferred embodiment, the lipid conjugate is PEG-lipid.Examples of PEG-lipid include but are not limited to PEG coupled to dialkyloxypropyl (PEG-DAA), for example, as described in PCT Publication No. 05 / 026372; PEG coupled to diacylglycerol (PEG-DAG), for example, as described in US Patent Application Publication No. 20030077829 and US Patent Application Publication No. 2005008689; PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE); PEG conjugated to ceramide, for example, as described 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.

[0437] PEG is a linear, water-soluble polymer of repeating ethylene glycol units. PEG is classified by its molecular weight; for example, PEG2000 has an average molecular weight of about 2,000 daltons, and PEG5000 has an average molecular weight of about 5,000 daltons. PEG is commercially available from Sigma Chemical Co. and other companies, and includes, for example, monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH2), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), and monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM). Other PEGs (e.g., mPEG (20KDa) amine), such as those described in U.S. Patent Nos. 6,774,180 and 7,053,150, are also useful for preparing the PEG-lipid conjugates of the present invention. The disclosures of these patents are incorporated herein by reference in their entirety for all purposes. Furthermore, monomethoxypolyethylene glycol acetic acid (MePEG-CH2COOH) is particularly useful for preparing PEG-lipid conjugates, including, for example, PEG-DAA conjugates.

[0438] 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.

[0439] In certain cases, PEG can be optionally substituted with alkyl, alkoxy, acyl or aryl groups.PEG can be directly conjugated to lipid, or can be linked to lipid via linker moiety.For example, any linker moiety suitable for coupling PEG to lipid can be used, including ester-free linker moiety and ester-containing linker moiety.In a preferred embodiment, the linker moiety is ester-free linker moiety.As used herein, the term "ester-free linker moiety" refers to a linker moiety that does not contain 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 PEG to a lipid.

[0440] 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 (-O-(O)POH-O-), sulfonate, and combinations thereof.

[0441] Additional PEG-lipid conjugates suitable for use in the present invention include, but are not limited to, compounds of the formula: ABC or a salt thereof, wherein 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 (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. alkoxy is substituted with one or more anion precursor groups, and wherein any of the (C-C)alkyl, (C-C)cycloalkyl, (C-C)cycloalkyl(C-C)alkyl, (C-C)alkoxy, (C-C)alkenyl, (C-C)alkynyl, (C-C)alkanoyl, (C-C)alkoxycarbonyl, (C-C)alkylthio, and (C-C)alkanoyloxy is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, (C-C)alkoxy, (C-C)alkanoyl, (C-C)alkoxycarbonyl, (C-C)alkylthio, or (C-C)alkanoyloxy; B is a polyethylene glycol chain having a molecular weight of about 550 daltons to about 10,000 daltons; C is -LR a and; L is a direct bond, -C(O)O-, or -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)CCHCHC(O)-, and -NHC(O)CHCHC(O)NH-; R a is a branched (C 10 -C50 ) alkyl or branched (C 10 -C 50 ) alkenyl, wherein said branched (C 10 -C 50 ) alkyl or branched (C 10 -C 50 ) one or more carbon atoms of the alkenyl are replaced with -O-; R b are each independently H or (C1-C6) alkyl.

[0442] The conjugated lipids may comprise, for example, PEG-lipids comprising compounds of the formula A-PEG-diacylglycerol (DAG), A-PEG-dialkyloxypropyl (DAA), A-PEG-phospholipid, A-PEG-ceramide (Cer), or mixtures thereof, wherein A is (C1-C6) alkyl, (C3-C8) cycloalkyl, (C3-C8) cycloalkyl (C1-C6) alkyl, (C1-C6) alkoxy, (C2-C6) alkoxy, or PEG-lipid. alkenyl, (C2-C6)alkynyl, (C1-C6)alkanoyl, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, or (C2-C6)alkanoyloxy, wherein any (C1-C6)alkyl, (C3-C8)cycloalkyl, (C3-C8)cycloalkyl(C1-C6)alkyl, (C1-C6)alkoxy, (C2-C6)alkenyl, (C2-C6)alkynyl, (C1-C 6) Alkanoyl, (C-C)alkoxycarbonyl, (C-C)alkylthio, and (C-C)alkanoyloxy are substituted with one or more anion precursor groups, and wherein any of (C-C)alkyl, (C-C)cycloalkyl, (C-C)cycloalkyl(C-C)alkyl, (C-C)alkoxy, (C-C)alkenyl, (C-C)alkynyl, (C The (1-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.

[0443] Phosphatidylethanolamines with various acyl chain groups of varying chain length and 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 to 100 are preferred. Phosphatidylethanolamines containing 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).

[0444] 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 herein by reference in their entireties 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; and 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 where R is a member selected from the group consisting of4 and R 5 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 skilled in the art that other polyamides can be used in the compounds of the present invention.

[0445] The term "diacylglycerol" refers to the two fatty acyl chains R 1 and R 2 The term "acyl group" refers to a compound having 2 to 30 carbon atoms, both of which are independently attached to the 1- and 2-positions of glycerol by ester bonds. The acyl group 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 icosyl (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] It has.

[0446] The term "dialkyloxypropyl" refers to two alkyl chains, R 1 and R 2 where 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] It has.

[0447] In a preferred embodiment, the PEG-lipid has the following formula: [ka] wherein R 1 and R 2 are independently selected 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 icosyl (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.

[0448] 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 preferred embodiments, 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 or methyl group.

[0449] In a preferred embodiment, "L" is an ester-free linker moiety. Suitable ester-free linkers include, but are not limited to, an amide linker moiety, an amino linker moiety, a carbonyl linker moiety, a carbamate linker moiety, a urea linker moiety, an ether linker moiety, a disulfide linker moiety, a succinamidyl linker moiety, 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).

[0450] In certain embodiments, the PEG-lipid conjugate is selected from the following: [ka]

[0451] In one embodiment, n is selected to provide a polymer chain with a molecular weight of about 2000.

[0452] 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. For example, see 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).

[0453] Preferably, the PEG-DAA conjugate is dilauryloxypropyl (C 12 )-PEG conjugate, dimyristyloxypropyl (C 14 )-PEG conjugate, dipalmityloxypropyl (C 16 )-PEG conjugate, or distearyloxypropyl (C 18 )-PEG conjugates. Those of skill in the art will readily appreciate that other dialkyloxypropyls can be used in the PEG-DAA conjugates of the present invention.

[0454] In addition to the above, it will be readily apparent to those skilled in the art that other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0455] The charge of the polycationic moiety can be distributed throughout the particle, or alternatively, can be a charge density of different concentrations in certain regions of the particle, e.g., a charge spike. When the charge density is distributed on the particle, the charge density can be uniformly or non-uniformly distributed. All variations in the charge distribution of the polycationic moiety are encompassed by the present invention.

[0456] In certain cases, the polycationic moiety can be conjugated with 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 conjugated. In certain cases, the conjugated ligand has a positive charge. Suitable ligands include compounds or devices with reactive functional groups, including, but not limited to, lipids, amphiphilic lipids, carrier compounds, biocompatible compounds, biomaterials, biopolymers, biomedical devices, analytically detectable compounds, therapeutically active compounds, enzymes, peptides, proteins, antibodies, immunostimulants, radiolabels, fluorophores, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, other targeting moieties, or toxins.

[0457] The lipid conjugate (e.g., PEG-lipid) typically comprises about 0.1 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 1 mol% to about 10 mol%, about 2 mol% to about 10 mol%, about 2 mol% to about 8 mol%, about 2 mol% to about 5 mol%, about 2 mol% to about 4 mol%, about 0.6 mol% to about 1.9 mol%, about 0.7 mol% to about 1.8 mol% of the total lipid present in the particle. ol%, 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%.

[0458] One of skill in the art will appreciate that the concentration of lipid complexes can be varied depending on the lipid complex employed and the rate at which the nucleic acid-lipid particles become fusogenic.

[0459] By controlling the composition and concentration of lipid complexes, it is possible to control the rate at which lipid complexes are exchanged with the outside of nucleic acid-lipid particles, and then the rate at which nucleic acid-lipid particles become membrane fusogenic.For example, when using PEG-phosphatidylethanolamine complexes or PEG-ceramide complexes as lipid complexes, the rate at which nucleic acid-lipid particles become membrane fusogenic can be changed, for example, by changing the concentration of lipid complexes, by changing the molecular weight of PEG, or by changing the chain length and saturation of the 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 particles become membrane fusogenic.Other methods that can be used to control the rate at which nucleic acid-lipid particles become membrane fusogenic will be clear to those skilled in the art after reading this disclosure.

[0460] Preparation of lipid particles 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, a continuous mixing method or a direct dilution process.

[0461] In a preferred embodiment, the non-cationic lipid is selected from the group consisting of 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-dielaidoyl-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.

[0462] In certain embodiments, the present invention provides LNPs produced via a continuous mixing process, e.g., a process comprising 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 herein by reference in its entirety for all purposes.

[0463] By successively introducing lipid and buffer solution into a mixing environment such as a mixing chamber, the lipid solution is successively diluted with the buffer solution, thereby producing liposomes virtually instantly upon mixing.As used herein, the phrase "successively diluting the lipid solution with the buffer solution" (and variations thereof) generally means that the lipid solution is diluted sufficiently quickly and with sufficient force to produce vesicles in the hydration process.By mixing the aqueous solution containing nucleic acid with the organic lipid solution, the organic lipid solution undergoes successive stepwise dilution in the presence of the buffer solution (i.e., aqueous solution), producing nucleic acid-lipid particles.

[0464] LNPs formed using continuous mixing methods typically have a size of about 40 nm to about 150 nm, about 40 nm to about 80 nm, about 40 nm to about 60 nm, about 50 nm to about 60 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm. The particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.

[0465] In another embodiment, the present invention provides LNPs produced by a direct dilution process, which involves forming a liposome solution and immediately introducing the liposome solution directly 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.

[0466] In yet another embodiment, the present invention provides LNPs produced by a direct dilution process, in which a third reservoir containing a dilution buffer is fluidly connected to a second mixing region. In this embodiment, the liposome solution formed in the first mixing region is immediately mixed directly with the dilution buffer in the second mixing region. In a preferred embodiment, the second mixing region includes a T-connector positioned so that the liposome solution and dilution buffer meet at a 180° angle, although connectors providing a shallower angle, 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 embodiment, the flow rate of the dilution buffer supplied to the second mixing region is controlled to be substantially equal to the flow rate of the liposome solution introduced thereto from the first mixing region. In another embodiment, the flow rate of the dilution buffer supplied to the second mixing region is controlled to be substantially different from 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 dilution buffer that is 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 smaller particle sizes at reduced concentrations.

[0467] These processes and equipment for carrying out these direct dilution processes are described in detail in U.S. Patent Publication No. 20070042031, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0468] LNPs formed using the direct dilution process typically have a size of about 40 nm to about 150 nm, about 40 nm to about 80 nm, about 40 nm to about 60 nm, about 50 nm to about 60 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm. The particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.

[0469] 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.

[0470] Several techniques are available for sizing particles to a desired size. One sizing method used for liposomes and equally applicable to the particles of the present invention is described in U.S. Patent No. 4,737,323, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Sonicating a particle suspension, either by bath sonication or probe sonication, results in a gradual reduction in size down to particles less than about 50 nm in size. Homogenization is another method that relies on shear energy to fragment larger particles into smaller ones. A typical homogenization procedure involves recirculating particles through a standard emulsion homogenizer until a selected particle size, usually about 60 to about 80 nm, is observed. In both methods, particle size distribution can be monitored by conventional laser-beam particle size discrimination or QELS.

[0471] Extrusion of particles through fine-porous 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 achieved. Particles can be extruded through successively smaller pore membranes to achieve stepwise size reduction.

[0472] 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 herein in its entirety for all purposes.

[0473] 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.

[0474] 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 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.

[0475] 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), or from about 4 (4:1). ) 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.

[0476] As mentioned above, the complex lipid may further comprise CPL. Various general methods for producing LNP-CPL (CPL-containing LNP) are discussed herein. Two general techniques include the "post-insertion" technique, i.e., inserting CPL into, for example, pre-formed LNP, and the "standard" technique, for example, including CPL in the lipid mixture during 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 produces 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 is also useful 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. WO00 / 62813, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0477] Other methods for making LNPs can be found, for example, in U.S. Patent No. 9,005,654 and PCT Publication No. WO2007 / 012191, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0478] kit The present invention also provides lipid particles (e.g., LNPs) in kit form. The kit may include compartmentalized containers for holding 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, along with instructions for its rehydration and administration.

[0479] As described herein, lipid particles (for example, LNP) of the present invention can be adapted to preferentially target specific tissues, organs, or tumors of interest.In certain cases, the 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 1:57PEG-cDSALNP formulation can be used to preferentially target tumors outside the liver, while 1:57PEG-cDMALNP formulation can be used to preferentially target the liver (including liver tumors).

[0480] In certain other cases, it may be desirable to have a targeting moiety 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.

[0481] Administration of lipid particles Once formed, 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. Accordingly, 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 allow delivery of the active or therapeutic agent to the cells.

[0482] The lipid particles (e.g., LNP) of the present invention can be adsorbed to almost any cell type by mixing or contacting them.Once adsorbed, the particles can be endocytosed by a portion of the cells, exchange lipids with the cell membrane, or fuse with the cell.The introduction or incorporation of the active agent or therapeutic agent (e.g., nucleic acid) portion of the particle can occur through any one of these routes.In particular, when fusion occurs, the particle membrane is incorporated into the cell membrane, and the particle contents are mixed with intracellular fluid.

[0483] The lipid particles (e.g., LNPs) of the present invention can be administered alone or in admixture with a pharmaceutically acceptable carrier (e.g., saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practice. Typically, buffered saline (e.g., 135-150 mM NaCl) will be used as the pharmaceutically 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, and globulins. Further suitable carriers are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans.

[0484] The pharmaceutically acceptable carrier is generally added after the lipid particles are formed, and thus, after the particles are formed, they can be diluted in a pharmaceutically acceptable carrier, such as ordinary buffered saline.

[0485] The concentration of particles in pharmaceutical formulations can vary significantly, i.e., less than about 0.05% by weight, typically about 2-5% by weight, to as high as about 10-90% by weight, and will be selected primarily based on fluid volume, viscosity, etc., in accordance with the particular mode of administration selected. For example, concentrations may be increased to reduce the fluid load associated with treatment. This may be particularly desirable for patients with atherosclerosis-related congestive heart failure or severe hypertension. Alternatively, particles composed of irritating lipids may be diluted to low concentrations to reduce inflammation at the administration site.

[0486] 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 pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, osmolality adjusters, 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 a lipid protectant to 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.

[0487] In vivo administration Systemic delivery for in vivo therapy, e.g., 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.

[0488] For in vivo administration, administration can be by any method known in the art, for example, injection, oral administration, inhalation (e.g., intranasal or intratracheal), transdermal application, or rectal administration. Administration can be in a single dose 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. Pat. 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 for administering lipid-based therapeutic agents are described in, for example, U.S. Patent Nos. 3,993,754, 4,145,410, 4,235,871, 4,224,179, 4,522,803, and 4,588,578. 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 above references are incorporated herein by reference in their entirety for all purposes.

[0489] 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., intranasal or intratracheal) (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, etc.

[0490] In certain embodiments, pharmaceutical compositions can be delivered by nasal spray, inhalation, and / or other aerosol delivery vehicles.Methods for directly delivering nucleic acid compositions to the lungs by nasal 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.

[0491] 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.

[0492] Generally, for intravenous administration, lipid particle formulations are formulated using a suitable pharmaceutical carrier. Many pharmaceutically acceptable carriers can be used in the compositions and methods of the present invention. Suitable formulations for use in the present invention can be found, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). Various aqueous carriers, such as water, buffered water, 0.4% saline, 0.3% glycine, etc., may be used, and these aqueous carriers may contain glycoproteins for enhanced stability, such as albumin, lipoproteins, globulins, etc. Buffered saline (135-150 mM NaCl) is commonly used as a pharmaceutically acceptable carrier, although other suitable carriers may also suffice. These compositions can be sterilized by conventional liposome sterilization techniques, such as filtration. The compositions may contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, osmolality adjusting agents, wetting agents, etc., required to approximate physiological conditions, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. These compositions may be sterilized using the techniques described above, or alternatively, may be produced under sterile conditions. The resulting aqueous solutions may be packaged for use or may be filtered under aseptic conditions and lyophilized, with the lyophilized preparation being combined with a sterile aqueous solution prior to administration.

[0493] In certain applications, the lipid particles disclosed herein can be delivered to individuals 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, wafers, 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 dosage unit 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 pharmaceutically pure and substantially non-toxic in the amounts employed.

[0494] 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 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 can be prepared in such a way that a suitable dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, administration route, product shelf life, and other pharmacological considerations are taken into account by those skilled in the art when preparing such pharmaceutical formulations, and therefore, various dosages and treatment regimens may be desirable.

[0495] 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) capsules, sachets, or tablets, each containing a predetermined amount of a therapeutic agent, such as a nucleic acid (e.g., interfering RNA or mRNA), as a liquid, solid, granules, or gelatin; (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 pharmaceutically compatible carriers. The lozenge form may include a pastille containing a 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, etc.

[0496] 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.

[0497] When preparing pharmaceutical preparations of the lipid particles of the present invention, it is preferable to use a large amount 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.

[0498] The methods of the present invention can be practiced in a variety of hosts. Preferred hosts include mammalian species such as primates (e.g., humans and chimpanzees, as well as other non-human primates), dogs, cats, horses, cows, sheep, goats, rodents (e.g., rats and mice), rabbits, and pigs.

[0499] 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 particles.

[0500] 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.

[0501] When performed in vitro, contact between cells and lipid particles occurs in a biologically compatible medium. The particle concentration varies widely depending on the specific application, but is generally about 1 μmol to about 10 mmol. Treatment of cells with lipid particles is generally carried out at physiological temperature (about 37°C) for a period of about 1 to 48 hours, preferably about 2 to 4 hours.

[0502] In one group of preferred embodiments, the lipid particle suspension is added to about 10 3 ~about 10 5 cells / ml, more preferably about 2 x 10 4The 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.

[0503] An endosomal release parameter (ERP) assay can be used to optimize the delivery efficiency of LNPs or other lipid particles of the present invention. The ERP assay is described in detail in U.S. Patent Publication No. 20030077829, the disclosure of which is incorporated herein by reference in its entirety for all purposes. More specifically, the 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 / uptake or fusion with / destabilization of endosomal membranes. This assay quantitatively determines how each component of an LNP or other lipid particle affects delivery efficiency, thereby enabling optimization of the LNP or other lipid particle. Typically, an ERP assay measures the expression of a reporter protein (e.g., luciferase, β-galactosidase, green fluorescent protein (GFP), etc.). In some cases, LNP formulations optimized for expression plasmids are also suitable for encapsulating interfering RNA or mRNA. In other cases, ERP assay can be adapted to measure the downregulation of the transcription or translation of target sequence in the presence or absence of interfering RNA (for example, 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 of each of various LNP or other lipid particles, it is possible to easily determine the optimized system, for example, the LNP or other lipid particle that is most effectively taken up into cells.

[0504] Cellular delivery of lipid particles The compositions and methods of the present invention are used to treat a wide variety of cell types in vivo and in vitro. Suitable cells include, for example, hematopoietic progenitor (stem) cells, fibroblasts, keratinocytes, hepatocytes, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, resting lymphocytes, terminally differentiated cells, slow-cycling or noncycling primary cells, parenchymal cells, lymphoid cells, epithelial cells, bone cells, etc. In a preferred embodiment, an active or therapeutic agent, such as one or more nucleic acid molecules (e.g., interfering RNA (e.g., siRNA) or mRNA) is delivered to cancer cells, such as, 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.

[0505] 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 present methods and compositions can be used in cells of a wide variety of vertebrates, including mammals such as dogs, cats, horses, cows, sheep, goats, rodents (e.g., mice, rats, and guinea pigs), rabbits, pigs, and primates (e.g., monkeys, chimpanzees, and humans).

[0506] To the extent that it may be required, cell tissue culture is well known in the art. For example, Freshney, Culture of Animal Cells, a Manual of Basic Technique, 3rd Ed., Wiley-Liss, New York (1994); Kuchler et al., Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977) and the references cited therein provide general guidance to cell culture. Cultured cell lines are often in the form of monolayer cells, although cell suspensions are also used.

[0507] 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 a therapeutic nucleic acid such as an interfering RNA (e.g., siRNA) or mRNA sequence, by detection of a target sequence of interest (i.e., by detecting expression or reduced expression of the sequence of interest), or a combination thereof.

[0508] Particle detection Lipid particles of the present invention, such as LNPs, can be detected using any method known in the art.For example, labels can be directly or indirectly coupled to components of lipid particles using methods 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 lipid particle components, stability requirements, and available measurement means and disposable equipment.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 These include, but are not limited to, radiolabels such as P; enzymes such as horseradish peroxidase, alkaline phosphatase, and spectrocolorimetric labels such as colloidal gold or colored glass or plastic beads such as polystyrene, polypropylene, latex, etc. The label can be detected using any means known in the art.

[0509] Nucleic acid detection Nucleic acid (for example, interfering RNA or mRNA) can be detected and quantified by any of several methods known to those skilled in the art.Nucleic acid detection can be carried out by well-known methods such as Southern analysis, Northern analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting and affinity chromatography.Additional biochemical analysis methods can also be used, such as spectrophotometry, X-ray imaging, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC) and hyperdiffusion chromatography.

[0510] The choice of nucleic acid hybridization format is not critical. Various 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).

[0511] 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 are known that are suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning.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™), are found in Sambrook et al., In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2000), and Ausubel et al., SHORT PROTOCOLS IN MOLECULAR BIOLOGY, eds., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (2002), as well as U.S. 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 in vitro cloning of 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 exhibit mutations. The disclosures of the above references are incorporated herein by reference in their entirety for all purposes.

[0512] For example, nucleic acids for use as probes in in vitro amplification methods, gene probes, or as inhibitor components are typically chemically synthesized according to the solid-phase phosphoramidite triester method described by Beaucage et al., Tetrahedron Letts., 22:1859-1862 (1981), using an automated synthesizer, for example, as described in Needham VanDevanter et al., Nucleic Acids Res., 12:6159 (1984). If necessary, purification of polynucleotides is typically performed by either native acrylamide gel electrophoresis or 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.

[0513] 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 in situ hybridization assays, cells are fixed on 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, allowing labeled specific probes to anneal. The probes are preferably labeled with radioisotopes or fluorescent reporters.

[0514] Ionizable lipids Ionizable cationic lipids are typically the primary lipid component in lipid nanoparticles (LNPs). They are designed to be charge-neutral at standard physiological pH (approximately 7), but acquire a positive charge at lower (acidic) pH. This aids in the encapsulation and intracellular delivery of nucleic acid payloads during LNP formation (the endosomal fusion process requires the positive charge of ionizable lipids).

[0515] Ionizable lipids such as MC3 (used in the FDA-approved siRNA-LNP product Onpattro) and 3D-P-DMA have demonstrated good activity and tolerability in a variety of applications. However, they are not rapidly metabolized and take several weeks to be cleared from target tissues. This makes them less suitable for applications requiring multiple administrations, especially those with short dosing intervals and / or chronic indications.

[0516] One way to achieve a more favorable profile is to include a carboxyl ester group in the hydrophobic domain of the lipid. Ester groups undergo enzymatic degradation in the body, which has been reported to improve lipid clearance in target tissues (Maier et al., 2013). However, the chemical structure immediately surrounding the ester must be considered, as it influences the efficacy of the strategy. ALC-0315, an ionizable lipid in BNT162b2 (also known as Pfizer's COVID vaccine), has a carboxylic ester in the hydrophobic domain but was found to be metabolized at a rate similar to that of MC3 [see Comirnaty evaluation report, European Medicines Agency]. Furthermore, it is necessary to incorporate esters while maintaining a favorable activity and tolerability profile.

[0517] To this end, a series of ionizable cationic lipids incorporating ester functional groups were designed, synthesized, and evaluated in the following manner. Compatibility (particle size, polydispersity, payload loading, pKa) Efficacy (mouse liver expression model, delivering luciferase-encoding mRNA and OTC mRNA) Tolerability (mouse immune stimulation model, cytokine readout) Lipid clearance (mouse model - clearance from target tissue (liver)).

[0518] The present invention will be described in more detail by specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present invention in any way. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to produce essentially the same results. [Example]

[0519] Preparation Example 1. Synthesis of heptadecane-9-ylidenehydrazine [ka] Step 1. Synthesis of 9-heptadecanol [ka] The Grignard reagent was prepared from 1-bromooctane (6 g, 31 mmol) and magnesium (808 mg, 33 mmol) in THF by stirring at 45 °C for 2 h. The reaction was cooled to 10 °C, and ethyl formate (2.6 mL, 32 mmol) in THF (5 mL) was added dropwise. HO (3 mL) and 6 M HCl (8 mL) were added to dissolve the residual magnesium. The reaction was partitioned between hexane and HO. The organics were washed with brine, dried (MgSO), filtered, and concentrated in vacuo. The residue was stirred in ethanol with a solution of KOH (1.6 g) in water (3 mL). The ethanol was removed in vacuo, and the residue was partitioned between 6 M HCl and hexane. The organics were washed with HO and brine, dried (MgSO), filtered, and concentrated in vacuo. The crude product was purified by automated flash chromatography (0-20% EtOAc / Hex) to give 9-heptadecanol (3.2 g, 81.2%).

[0520] Step 2.9-Synthesis of heptadecanone [ka] 9-Heptadecanol (3.2 g, 12.6 mmol) and PCC-SiO (1:1) (16.3 g, 38 mmol) were stirred in DCM (100 mL) at room temperature for 16 h. The reaction was filtered through silica and eluted with EtOAc. The organics were concentrated in vacuo and purified by automated flash chromatography (0–10% EtOAc / Hex) to give 9-heptadecanone (3.2 g, 99.6%).

[0521] Step 3. Synthesis of N'-(heptadecan-9-ylidene)tert-butoxycarbohydrazide [ka] tert-Butoxycarbohydrazide (195 mg, 1.47 mmol), 9-heptadecanone (750 mg, 2.9 mmol), and AcOH (catalytic) were stirred at reflux in MeOH (10 mL) for 15 h. The reaction was concentrated in vacuo and purified by automated flash chromatography (0–20% EtOAc / Hex) to give N'-(heptadecan-9-ylidene)tert-butoxycarbohydrazide (320 mg, 58.9%).

[0522] Step 4. Synthesis of heptadecane-9-ylidenehydrazine [ka] N'-(heptadecan-9-ylidene)tert-butoxycarbohydrazide (320 mg, 0.9 mmol) was stirred in 25% TFA / DCM (40 mL) at room temperature until the reaction was complete. The solvent was removed in vacuo to give heptadecan-9-ylidenehydrazine (231 mg, 99.1%), which was used without further purification.

[0523] Preparation Example 2. Synthesis of ((6Z,15Z)-henicosa-6,15-dien-11-ylidene)hydrazine [ka] Using a similar method as above, ((6Z,15Z)-henicosa-6,15-dien-11-ylidene)hydrazine was prepared.

[0524] Preparation Example 3. Synthesis of heptadecan-9-ylhydrazine [ka] Step 1. Synthesis of N'-(heptadecan-9-yl)tert-butoxycarbohydrazide [ka] tert-Butoxycarbohydrazide (412 mg, 3.1 mmol), 9-heptadecanone (794 mg, 3.1 mmol), AcOH (0.75 mL, 3.2 mmol), and NaBH(OAc) (2.0 g, 9.4 mmol) in DCM (20 mL) were stirred at room temperature for 2 days and at 40 °C for an additional 20 h. The reaction was washed with saturated NaHCO and brine, dried (MgSO), filtered, and concentrated in vacuo. The residue was purified by automated flash chromatography (0–12% EtOAc / Hex) to give N′-(heptadecan-9-yl)tert-butoxycarbohydrazide (700 mg, 60.5%).

[0525] Step 2. Synthesis of heptadecan-9-ylhydrazine [ka] N'-(heptadecan-9-yl)tert-butoxycarbohydrazide (700 mg, 1.9 mmol) was stirred with 25% TFA in DCM (10 mL) at room temperature for 1.5 hours, and the reaction was concentrated in vacuo to give heptadecan-9-ylhydrazine (500 mg, 97.9%), which was used without purification.

[0526] Preparation Example 4. Synthesis of ((6Z,15Z)-henicosa-6,15-dien-11-yl)hydrazine Using a similar method as above, ((6Z,15Z)-henicosa-6,15-dien-11-yl)hydrazine was prepared. [ka]

[0527] Preparative Example 5. Synthesis of 1,1-dioctylhydrazine [ka] Step 1. Synthesis of N',N'-dioctyl tert-butoxycarbohydrazide [ka] Octanal (2.4 mL, 15.0 mmol), tert-butoxycarbohydrazide (1 g, 7.6 mmol), and AcOH (7.6 mmol) were stirred in anhydrous MeOH under a nitrogen atmosphere at 75 °C for 1 h. After cooling to room temperature, NaBHCN (1.9 g, 30.3 mmol) was added, and the mixture was stirred at room temperature for 15 h. The solvent was removed in vacuo, and the residue was dissolved in EtOAc, washed with HO and brine, dried (MgSO), filtered, and concentrated. The crude material was purified by automated flash chromatography (0–10% EtOAc / Hex) to give N′,N′-dioctyl tert-butoxycarbohydrazide (2.0 g, 74.3%).

[0528] Step 2. Synthesis of 1,1-dioctylhydrazine [ka] N',N'-Dioctyl tert-butoxycarbohydrazide (1.1 g, 3.0 mmol) was stirred in 30% TFA in DCM (20 mL) at 0°C until the reaction was complete and allowed to warm to room temperature. The solvent was removed in vacuo to give 1,1-dioctylhydrazine (782 mg, Quant), which was used without purification.

[0529] Preparative Example 6. Synthesis of 1,1-di((Z)-dec-4-en-1-yl)hydrazine [ka] A similar method was used to prepare 1,1-di((Z)-dec-4-en-1-yl)hydrazine.

[0530] Preparative Example 7. General Method (a) for the Synthesis of Branched Alkyl Alcohols: Illustrated by the Synthesis of 5-Octyltridec-4-en-1-ol Step 1. Synthesis of 9-(4-bromobutyl)heptadecan-9-ol [ka] The Grignard reagent was prepared from Mg (2.6 g, 105 mmol) and 1-bromooctane (20.0 g, 105 mmol) in anhydrous Et2O (35 mL) at reflux for 1 h. The reaction was cooled, and a solution of ethyl 5-bromopentanoate (8.0 g, 38.3 mmol) in anhydrous Et2O (25 mL) was added dropwise, followed by an additional 1 h of reflux. The reaction was cooled to room temperature and poured into 100 mL of saturated ice-cold NH4Cl solution. The aqueous portion was extracted with Et2O, and the organics were dried (MgSO4), filtered, and concentrated in vacuo. The crude material was purified by automated flash chromatography (0–20% EtOAc / Hex) to give 9-(4-bromobutyl)heptadecan-9-ol (10.6 g, 70.7%).

[0531] Step 2. Synthesis of 9-(4-bromobutylidene)heptadecane [ka] 9-(4-Bromobutyl)heptadecan-9-ol (10.6 g, 27.0 mmol) and p-TsOH.HO (93 mg, 0.54 mmol) were stirred under N at 130 °C for 2 h. The crude material was purified by automated flash chromatography (0–4% EtOAc / Hex) to give 9-(4-bromobutylidene)heptadecane (8.84 g, 87.5%).

[0532] Step 3. Synthesis of 5-octyltridec-4-en-1-ol [ka] 9-(4-Bromobutylidene)heptadecane (8.84 g, 23.7 mmol), benzyl(chloro)triethylamine (540 mg, 2.4 mmol), and NaOAc (7.8 g, 94.7 mmol) in DMF (60 mL) were stirred at 70 °C for 20 h. The reaction was poured into ice / water and extracted with EtO. The combined organics were dried (MgSO), filtered, and concentrated in vacuo. The residue was dissolved in MeOH, KOH (18.6 g, 332 mmol) in HO was added, and the reaction was stirred for 30 min. The MeOH was removed in vacuo, and the reaction was partitioned between HO and EtO. The combined organics were dried (MgSO4), filtered, concentrated in vacuo and the crude material was purified by automated flash chromatography (0-15% EtOAc / Hex) to give 5-octyltridec-4-en-1-ol (4.5 g, 60.8%).

[0533] Step 4. Synthesis of 5-octyltridecan-1-ol [ka] 5-Octyltridec-4-en-1-ol (4.469 g, 14.39 mmol) was subjected to catalytic hydrogenation over Pd—C in MeOH (50 mL) at room temperature for 15 hours. The reaction was filtered and concentrated in vacuo to give 5-octyltridecan-1-ol (3.5 g, 77.8%), which was used without further purification.

[0534] The following compounds were prepared using methods similar to those described above. [ka]

[0535] Preparative Example 8. General Method for the Synthesis of Branched Alkyl Alcohols (b): Illustrated by the Synthesis of 3-Pentyloctan-1-ol Step 1. Synthesis of ethyl 3-pentyloct-2-enoate [ka] Triethyl phosphonoacetate (65.8 g, 294 mmol) was stirred in THF (220 mL) at −10° C. 1 M LiHMDS in THF (294 mL, 294 mmol) was added dropwise and the reaction was stirred at −10° C. for 1 h, followed by 1 h at 0° C. 6-Undecanone (25 g, 147 mmol) was added and the reaction was stirred at 45° C. for 16 h. The reaction was quenched with HO, extracted with EtO, and washed with brine. The organics were dried (MgSO), filtered, and concentrated in vacuo to give ethyl 3-pentyloct-2-enoate (43.5 g), which was used in the next step without further treatment.

[0536] Step 2.3-Synthesis of ethyl pentyl octanoate [ka] Ethyl 3-pentyloct-2-enoate (35 g, 147 mmol) was subjected to catalytic hydrogenation over Pd—C in MeOH (100 mL) at room temperature for 20 hours. The reaction was filtered and concentrated in vacuo to give ethyl 3-pentyloctanoate (33.3 g, 93.6%), which was used without further purification.

[0537] Step 3. Synthesis of 3-pentyloctan-1-ol [ka] LiAlH4 (6.8 g, 179 mmol) was stirred in THF (500 mL) at 0 °C for 30 min. Ethyl 3-pentyloctanoate (33.3 g, 138 mmol in THF (500 mL)) was added dropwise and the reaction was stirred at room temperature until complete. The reaction was cooled to 0 °C, diluted with Et2O (500 mL), and quenched with 1 M NaOH (16 mL). The reaction was stirred for 30 min, dried (MgSO4), filtered through Celite, and concentrated in vacuo. The crude material was purified by automated flash chromatography (0–30% EtOAc / Hex) to give 3-pentyloctan-1-ol (24.7 g, 89.7%).

[0538] The following compounds were prepared using methods similar to those described above. [ka]

[0539] Preparation Example 9. Synthesis of (3,5-dihexylphenyl)methanol Step 1. Synthesis of methyl 3,5-bis(hex-1-yn-1-yl)benzoate [ka] Methyl 3,5-dibromobenzoate (7.4 g, 25.0 mmol), hexyne (8.2 g, 100.0 mmol), dichlorobis[4-(diphenylphosphanyl)phenyl]palladium (1.4 g, 2.0 mmol), CuI (0.2 g, 1.0 mmol), and TEA (21.0 mL, 150 mmol) in benzene (100 mL) were stirred at 80 °C for 48 h. The reaction was filtered through a pad of silica and eluted with EtOAc. The organics were concentrated in vacuo, and the crude material was purified by automated flash chromatography (0–20% EtOAc / Hex) to give methyl 3,5-bis(hex-1-yn-1-yl)benzoate (7.0 g, 94.4%).

[0540] Step 2. Synthesis of methyl 3,5-dihexylbenzoate [ka] Methyl 3,5-bis(hex-1-yn-1-yl)benzoate (7.0 g, 23.6 mmol) was subjected to catalytic hydrogenation over Pd—C in EtOH (100 mL) at room temperature for 18 hours. The reaction was filtered through Celite and concentrated in vacuo to give methyl 3,5-dihexylbenzoate (6.3 g, 87.7%), which was used without further purification.

[0541] Step 3. Synthesis of (3,5-dihexylphenyl)methanol [ka] Methyl 3,5-dihexylbenzoate (6.3 g, 20.7 mmol) was stirred in anhydrous THF at 0 °C. LiAlH (1.2 g, 31.0 mmol) was added portionwise and the reaction stirred for 1 h, allowing it to warm to room temperature. Upon completion, the reaction was quenched with HO and 1 M NaOH and extracted with EtOAc. The organics were dried (MgSO), filtered, concentrated in vacuo, and purified by automated flash chromatography (10% EtOAc / Hex) to give (3,5-dihexylphenyl)methanol (3.0 g, 52.4%).

[0542] Preparative Example 10. Synthesis of 2-fluoro-2-hexyldecanoic acid Step 1. Synthesis of ethyl 2-hexyldecanoate [ka] Hexyldecanoic acid (3 g, 11.7 mmol) and H2SO4 (catalyst) were refluxed in EtOH (20 mL) for 15 h. The reaction was concentrated in vacuo, and the residue was partitioned between hexane and water. The organics were dried (MgSO4), filtered, and concentrated in vacuo, and the residue was purified by automated flash chromatography (0-6% EtOAc / Hex) to give ethyl 2-hexyldecanoate (3.0 g, 90.7%).

[0543] Step 2. Synthesis of ethyl 2-fluoro-2-hexyldecanoate [ka] Ethyl 2-hexyldecanoate (1 g, 3.5 mmol) in THF was added dropwise to a solution of LDA (2.6 mL, 2 M, 5.273 mmol) at -78 °C and stirred for 0.5 h. N-Fluorobenzenesulfonimide (1.7 g, 5.3 mmol) in THF (15 mL) was added dropwise, and the reaction was stirred for 18 h while warming to room temperature. The reaction was diluted with EtOAc, washed sequentially with NH4Cl, water, and brine, dried (MgSO4), concentrated in vacuo, and purified by automated flash chromatography (0–6% EtOAc / Hex). NMR indicated 60% conversion. The reaction was repeated once more to give ethyl 2-fluoro-2-hexyldecanoate (770 mg, 72.4%).

[0544] Step 3. Synthesis of 2-fluoro-2-hexyldecanoic acid [ka] Ethyl 2-fluoro-2-hexyldecanoate (770 mg, 2.5 mmol) and LiOH (125 mg, 5.2 mmol) were stirred in MeOH (10 mL) and HO (2 mL) at room temperature for 15 h. 6 M HCl (0.87 mL) was added and MeOH was removed in vacuo. The residue was dissolved in DCM, washed with brine, dried (MgSO), filtered, and concentrated in vacuo to give 2-fluoro-2-hexyldecanoic acid (520 mg, 74.4%).

[0545] Preparative Example 11. Synthesis of Amino Alcohol THP Ethers, Illustrated by the Synthesis of 5-(Oxan-2-yloxy)pentan-1-amine Step 1. Synthesis of benzyl N-(5-hydroxypentyl)carbamate [ka] 5-Aminopentanol (21.0 g, 203 mmol) and benzyl 2,5-dioxopyrrolidin-1-yl carbonate (50.7 g, 203 mmol) were stirred in THF at room temperature for 16 hours. The reaction was concentrated in vacuo, and the residue was dissolved in EtOAc (250 mL), washed with 1 M HCl and 0.5 M NaOH, dried (MgSO), filtered, and concentrated in vacuo. The resulting oil was stirred in hexane until the product crystallized. The solid was collected by filtration to give benzyl N-(5-hydroxypentyl)carbamate (44 g, 91.0%).

[0546] Step 2. Synthesis of benzyl N-[5-(oxan-2-yloxy)pentyl]carbamate Benzyl N-(5-hydroxypentyl)carbamate (20.0 g, 84 mmol), 2H-pyran, 3,4-dihydro- (8.6 g, 101) and pTsOH-H2O (1.6 g, 8.4 mmol) were stirred in DCM (270 mL) at room temperature for 2 h. [ka] Benzyl N-(5-hydroxypentyl)carbamate (20.0 g, 84 mmol), 2H-pyran, 3,4-dihydro- (8.6 g, 101), and pTsOH-HO (1.6 g, 8.4 mmol) were stirred in DCM (270 mL) at room temperature for 2 h. The reaction was washed with NaHCO (saturated solution) and brine, dried (MgSO), filtered, and concentrated in vacuo. The crude material was purified by automated flash chromatography (10–50% EtOAc / Hex) to give benzyl N-[5-(oxan-2-yloxy)pentyl]carbamate (19.5 g, 72.0%).

[0547] Step 3. Synthesis of 5-(oxan-2-yloxy)pentan-1-amine [ka] Benzyl N-[5-(oxan-2-yloxy)pentyl]carbamate (19.0 g, 59 mmol) was subjected to catalytic hydrogenation over Pd / C in MeOH at room temperature for 16 h. The reaction was filtered through Celite and concentrated in vacuo to give 5-(oxan-2-yloxy)pentan-1-amine (10.9 g, 98.5%), which was used without purification.

[0548] The following compounds...

Claims

1. A compound of formula (I) or a salt thereof: 【Chemistry 1】 [In the formula, X is -CH ２ -, -O-, -O(C=O), or -C(=O)-; Y is absent or -CH ２ -, -O-, -SO ２ -, or -C(=O)-; R １ is C ２ ~C ２５ is hydrocarbyl; R ２ is C １ ~C ３ alkyl, R ３ is C １ ~C ３ alkyl; or R ２ and R ３ together with the nitrogen to which they are attached form an aziridino, azetidino, or pyrrolidino ring; m is 0, 1, 2, 3, 4, 5, or 6; n is 0 to 15; o is 0 to 15; L １ is absent or is -(C=O)-O-, -(C=O)N(H)N(R ４ )-,-(C=O)N(H)-N=C(R ４ )-,-(C=O)N(H)N(H)C(R ４ ) (R ５ )-,-(C=O)-O-CH ２ -R ｚ or -O(C=O)-R ｗ - and L ２ is absent or is -(C=O)-O-, -(C=O)N(H)N(R ４’ )-,-(C=O)N(H)-N=C(R ４’ )-,-(C=O)N(H)N(H)C(R ４’ ) (R ５’ )-,-(C=O)-O-CH ２ -R ｚ’ or -O(C=O)-R ｘ - and a is H or F; b is H or F; c is H or F and d is H or F; or c and d together with the carbon to which they are attached form -C(=O)-; R ４ is H or C ２ ~C ２５ is hydrocarbyl; R ５ is H or C ２ ~C ２５ is hydrocarbyl; R ４’ is C ２ ~C ２５ is hydrocarbyl; R ５’ is H or C ２ ~C ２５ is hydrocarbyl; R ６ is C ２ ~C ２５ is hydrocarbyl; R ７ is C ２ ~C ２５ is hydrocarbyl; R ｗ is absent or is -C(F)H-, or -C(F) ２ - and R ｘ is absent or is -C(F)H-, or -C(F) ２ - and R ｚ is C ２ ~C １０ phenyl substituted with 1, 2, or 3 groups independently selected from hydrocarbyl; and R ｚ’ is C ２ ~C １０ and phenyl substituted with 1, 2, or 3 groups independently selected from hydrocarbyl.

2. X is -CH ２ The compound or salt according to claim 1, wherein

3. 2. The compound or salt of claim 1, wherein X is -C(=O)-.

4. The compound or salt of claim 1, wherein X is -O-.

5. R ２ The compound or salt according to any one of claims 1 to 4, wherein is methyl.

6. R ２ The compound or salt according to any one of claims 1 to 4, wherein is ethyl.

7. R ２ The compound or salt according to any one of claims 1 to 4, wherein is propyl.

8. R ３ The compound or salt according to any one of claims 1 to 7, wherein is methyl.

9. R ３ The compound or salt according to any one of claims 1 to 7, wherein is ethyl.

10. R ３ The compound or salt according to any one of claims 1 to 7, wherein is propyl.

11. The compound or salt according to any one of claims 1 to 10, wherein m is 1.

12. The compound or salt according to any one of claims 1 to 10, wherein m is 2.

13. The compound or salt according to any one of claims 1 to 10, wherein m is 3.

14. 【Chemical 2】 が、(CH ３ ) ２ P ２ CH ２ C(=O)-、(CH ３ ) ２ P ２ CH ２ CH ２ C(=O)-、(CH ３ ) ２ P ２ CH ２ CH ２ CH ２ C(=O)-、(CH ３ ) ２ P ２ CH ２ -、 (CH ３ ) ２ NCH ２ CH ２ CH-, 5-pyrrolidin-1-ylpentanoyl, and (CH ３ ) ２ NCH ２ CH ２ CH ２ The compound of claim 1, wherein the compound is selected from the group consisting of: O-.

15. R １ C ５ ~C ２５ 15. A compound or salt according to any one of claims 1 to 14 which is hydrocarbyl.

16. R １ C ２ ~C ２５ The compound or salt according to any one of claims 1 to 14, which is alkyl.

17. R １ C ５ ~C ２５ The compound or salt according to any one of claims 1 to 14, which is alkyl.

18. R １ C ５ ~C ２５ 15. The compound or salt of any one of claims 1 to 14, which is alkenyl.

19. R １ The compound or salt according to any one of claims 1 to 14, wherein is 1-nonyl or 1-decyl.

20. 20. The compound or salt according to any one of claims 1 to 19, wherein o is 2 to 15.

21. 20. The compound or salt of any one of claims 1 to 19, wherein o is 10 to 15.

22. 20. The compound or salt according to any one of claims 1 to 19, wherein o is 2 to 10.

23. 20. The compound or salt according to any one of claims 1 to 19, wherein o is 2 to 5.

24. L ２ is -(C=O)-O-, -(C=O)N(H)N(R ４ ) (R ５ )-,-(C=O)N(H)-N=C(R ４ )-,-(C=O)N(H)N(H)C(R ４ ) (R ５ )-, or -(C=O)-O-CH ２ -R ｚ The compound according to any one of claims 1 to 23,

25. L ２ The compound or salt according to any one of claims 1 to 23, wherein is -(C=O)-O-.

26. L ２ -(C=O)N(H)N(R ４ ) (R ５ 24. The compound or salt according to any one of claims 1 to 23, wherein

27. L ２ -(C=O)-O-CH ２ -R ｚ The compound or salt according to any one of claims 1 to 23,

28. 28. The compound or salt of any one of claims 1 to 27, wherein a is H.

29. 28. The compound or salt according to any one of claims 1 to 27, wherein a is F.

30. The compound according to any one of claims 1 to 29, wherein b is H.

31. 30. The compound or salt according to any one of claims 1 to 29, wherein b is F.

32. R ６ C ２ ~C ２５ 32. A compound or salt according to any one of claims 1 to 31 which is hydrocarbyl.

33. R ６ C ５ ~C ２５ 32. A compound or salt according to any one of claims 1 to 31 which is hydrocarbyl.

34. R ６ C ２ ~C ２５ 32. The compound or salt of any one of claims 1 to 31, which is alkyl.

35. R ６ C ５ ~C ２５ 32. The compound or salt of any one of claims 1 to 31, which is alkyl.

36. R ６ C ５ ~C ２５ 32. The compound or salt of any one of claims 1 to 31, which is alkenyl.

37. 【Chemical 3】 but, 【Chemistry 4】 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 The compound or salt according to any one of claims 1 to 14, selected from the group consisting of:

38. 【Chemical 8】 but, 【Chemistry 9】 【Chemistry 10】 The compound or salt according to any one of claims 1 to 14, selected from the group consisting of:

39. 【Chemical 11】 but, 【Chemistry 12】 The compound or salt according to any one of claims 1 to 14, selected from the group consisting of:

40. Y is -CH ２ The compound or salt according to any one of claims 1 to 39, wherein

41. 40. The compound or salt of any one of claims 1 to 39, wherein Y is -C(=O)-.

42. 40. The compound or salt of any one of claims 1 to 39, wherein Y is -O-.

43. 43. The compound or salt according to any one of claims 1 to 42, wherein n is 2 to 15.

44. 43. The compound or salt according to any one of claims 1 to 42, wherein n is 10 to 15.

45. 43. The compound or salt according to any one of claims 1 to 42, wherein n is 2 to 10.

46. 43. The compound or salt according to any one of claims 1 to 42, wherein n is 2 to 5.

47. L １ is not present; and L ２ is -(C=O)-O-, -(C=O)N(H)N(R ４ ) (R ５ )-,-(C=O)N(H)-N=C(R ４ )-,-(C=O)N(H)N(H)C(R ４ ) (R ５ )-, or -(C=O)-O-CH ２ -R ｚ The compound according to any one of claims 1 to 46,

48. L １ is -(C=O)-O-, -(C=O)N(H)N(R ４ ) (R ５ )-,-(C=O)N(H)-N=C(R ４ )-,-(C=O)N(H)N(H)C(R ４ ) (R ５ )-, or -(C=O)-O-CH ２ -R ａ and L ２ The compound according to any one of claims 1 to 46, wherein is absent.

49. L １ is -(C=O)-O-, -(C=O)N(H)N(R ４ ) (R ５ )-,-(C=O)N(H)-N=C(R ４ )-,-(C=O)N(H)N(H)C(R ４ ) (R ５ )-, or -(C=O)-O-CH ２ -R ａ The compound according to any one of claims 1 to 46,

50. L １ The compound or salt according to any one of claims 1 to 46, wherein is -(C=O)-O.

51. L １ -(C=O)N(H)N(R ４ ) (R ５ 47. The compound or salt according to any one of claims 1 to 46, wherein

52. L １ -(C=O)-O-CH ２ -R ｚ 47. The compound or salt according to any one of claims 1 to 46,

53. 53. The compound or salt of any one of claims 1 to 52, wherein c is H.

54. 53. The compound or salt according to any one of claims 1 to 52, wherein c is F.

55. 55. The compound or salt of any one of claims 1 to 54, wherein d is H.

56. 55. The compound or salt of any one of claims 1 to 54, wherein d is F.

57. R ７ C ２ ~C ２５ 57. A compound or salt according to any one of claims 1 to 56 which is hydrocarbyl.

58. R ７ C ５ ~C ２５ 57. A compound or salt according to any one of claims 1 to 56 which is hydrocarbyl.

59. R ７ C ２ ~C ２５ 57. The compound or salt of any one of claims 1 to 56, which is alkyl.

60. R ７ C ５ ~C ２５ 57. The compound or salt of any one of claims 1 to 56, which is alkyl.

61. R ７ C ５ ~C ２５ 57. The compound or salt of any one of claims 1 to 56, which is alkenyl.

62. 【Catalog 13】 but, 【Chemistry 14】 【Chemistry 15】 40. The compound or salt of any one of claims 1 to 39, selected from the group consisting of:

63. 【Catalog 16】 but, 【Chemistry 17】 40. The compound or salt of any one of claims 1 to 39, selected from the group consisting of:

64. 【Catalog 18】 but, 【Chemistry 19】 38. The compound or salt according to any one of claims 1 to 37, selected from the group consisting of:

65. R ４ C ２ ~C ２５ 62. A compound or salt according to any one of claims 1 to 46 and 53 to 61 which is hydrocarbyl.

66. R ４ C ５ ~C ２５ 62. A compound or salt according to any one of claims 1 to 46 and 53 to 61 which is hydrocarbyl.

67. R ４ C ２ ~C ２５ 62. The compound or salt according to any one of claims 1 to 46 and 53 to 61, which is alkyl.

68. R ４ C ５ ~C ２５ 62. The compound or salt according to any one of claims 1 to 46 and 53 to 61, which is alkyl.

69. R ４ C ５ ~C ２５ 62. The compound or salt of any one of claims 1 to 46 and 53 to 61, which is alkenyl.

70. R ５ C ２ ~C ２５ 62. A compound or salt according to any one of claims 1 to 46 and 53 to 61 which is hydrocarbyl.

71. R ５ C ５ ~C ２５ 62. A compound or salt according to any one of claims 1 to 46 and 53 to 61 which is hydrocarbyl.

72. R ５ C ２ ~C ２５ 62. The compound or salt according to any one of claims 1 to 46 and 53 to 61, which is alkyl.

73. R ５ C ５ ~C ２５ 62. The compound or salt according to any one of claims 1 to 46 and 53 to 61, which is alkyl.

74. R ５ C ５ ~C ２５ 62. The compound or salt of any one of claims 1 to 46 and 53 to 61, which is alkenyl.

75. R ４’ C ２ ~C ２５ 37. A compound or salt according to any one of claims 1 to 23 and 28 to 36 which is hydrocarbyl.

76. R ４’ C ５ ~C ２５ 37. A compound or salt according to any one of claims 1 to 23 and 28 to 36 which is hydrocarbyl.

77. R ４’ C ２ ~C ２５ 37. The compound or salt according to any one of claims 1 to 23 and 28 to 36, which is alkyl.

78. R ４’ C ５ ~C ２５ 37. The compound or salt according to any one of claims 1 to 23 and 28 to 36, which is alkyl.

79. R ４’ C ５ ~C ２５ 37. The compound or salt according to any one of claims 1 to 23 and 28 to 36, which is alkenyl.

80. R ５’ C ２ ~C ２５ 37. A compound or salt according to any one of claims 1 to 23 and 28 to 36 which is hydrocarbyl.

81. R ５’ C ５ ~C ２５ 37. A compound or salt according to any one of claims 1 to 23 and 28 to 36 which is hydrocarbyl.

82. R ５’ C ２ ~C ２５ 37. The compound or salt according to any one of claims 1 to 23 and 28 to 36, which is alkyl.

83. R ５’ C ５ ~C ２５ 37. The compound or salt according to any one of claims 1 to 23 and 28 to 36, which is alkyl.

84. R ５’ C ５ ~C ２５ 37. The compound or salt according to any one of claims 1 to 23 and 28 to 36, which is alkenyl.

85. R ｚ But, C ２ ~C １０ 62. A compound or salt according to any one of claims 1 to 46 and 53 to 61, which is phenyl substituted with 1 or 2 groups independently selected from hydrocarbyl.

86. Each C ２ ~C １０ Hydrocarbyl is C ５ ~C １０ 86. The compound or salt of claim 85, independently selected from the group consisting of hydrocarbyls.

87. Each C ２ ~C １０ Hydrocarbyl is C ２ ~C １０ 86. The compound or salt of claim 85, wherein said compound or salt is independently selected from the group consisting of alkyl.

88. Each C ２ ~C １０ Hydrocarbyl is C ５ ~C １０ 86. The compound or salt of claim 85, wherein said compound or salt is independently selected from the group consisting of alkyl.

89. Each C ２ ~C １０ Hydrocarbyl is C ５ ~C １０ 86. The compound or salt of claim 85, independently selected from the group consisting of alkenyl.

90. R ｚ’ But, C ２ ~C １０ 37. A compound or salt according to any one of claims 1 to 23 and 28 to 36, which is phenyl substituted with 1 or 2 groups independently selected from hydrocarbyl.

91. Each C ２ ~C １０ Hydrocarbyl is C ５ ~C １０ 91. The compound or salt of claim 90, independently selected from the group consisting of hydrocarbyls.

92. Each C ２ ~C １０ Hydrocarbyl is C ２ ~C １０ 91. The compound or salt of claim 90, independently selected from the group consisting of alkyl.

93. Each C ２ ~C １０ Hydrocarbyl is C ５ ~C １０ 91. The compound or salt of claim 90, independently selected from the group consisting of alkyl.

94. Each C ２ ~C １０ Hydrocarbyl is C ５ ~C １０ 91. The compound or salt of claim 90, independently selected from the group consisting of: alkenyl.

95. 【Catalogue 20】 【Chemistry 21】 【Chemical 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 【Chemical 27】 【Chemistry 28】 【Chemical Formula 29】 【Chemistry 30】 【Chemistry 31】 【Chemistry 32】 【Chemical 33】 【Chemical 34】 【Chemistry 35】 【Chemical 36】 【Chemical 37】 【Chemical Formula 38】 【Chemical Formula 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 【Chemistry 43】 【Chemistry 44】 【Chemistry 45】 【Chemistry 46】 【Chemistry 47】 or a salt thereof.

96. 【Catalogue 48】 2. The compound or salt of claim 1, which is:

97. 【Catalogue 49】 2. The compound or salt of claim 1, which is:

98. 【Chemical 50】 2. The compound or salt of claim 1, which is:

99. 【Chemical 51】 2. The compound or salt of claim 1, which is: 【Request 100】 【Chemical 52】 2. The compound or salt of claim 1, which is:

101. 【Chemical 53】 2. The compound or salt of claim 1, which is:

102. 【Chemical 54】 2. The compound or salt of claim 1, which is:

103. 【Chemical 55】 2. The compound or salt of claim 1, which is:

104. 1. A nucleic acid-lipid particle comprising: (a) one or more nucleic acid molecules; (b) a non-cationic lipid; (c) complex lipids; and (d) A compound or salt according to any one of claims 1 to 103. wherein the one or more nucleic acid molecules are encapsulated within the lipid particle.

105. The nucleic acid-lipid particle of claim 104, wherein said compound or salt constitutes from about 30 mol% to about 85 mol% of the total lipid present in said particle; said non-cationic lipid constitutes from about 13 mol% to about 49.5 mol% of the total lipid present in said particle; and said complex lipid constitutes from about 0.1 mol% to about 10 mol% of the total lipid present in said particle.

106. The nucleic acid-lipid particle of claim 104, wherein said compound or salt constitutes from about 30 mol% to about 85 mol% of the total lipid present in said particle; said non-cationic lipid constitutes from about 13 mol% to about 49.5 mol% of the total lipid present in said particle; and said complex lipid constitutes from about 0.1 mol% to about 10 mol% of the total lipid present in said particle.

107. The nucleic acid-lipid particle of any one of claims 104 to 106, 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.

108. The nucleic acid-lipid particle of claim 107, wherein the nucleic acid is an mRNA molecule.

109. The nucleic acid-lipid particle of claim 107, wherein said nucleic acid comprises a double-stranded siRNA molecule.

110. The nucleic acid-lipid particle of claim 109, wherein said double-stranded siRNA molecule comprises at least one modified nucleotide.

111. The nucleic acid-lipid particle of claim 110, wherein said siRNA comprises at least one 2'-O-methyl (2'OMe) nucleotide.

112. The nucleic acid-lipid particle of any one of claims 104-111, wherein the non-cationic lipid comprises cholesterol or a derivative thereof.

113. 113. The nucleic acid-lipid particle of claim 112, wherein said cholesterol or a derivative thereof constitutes from about 31.5 mol % to about 42.5 mol % of the total lipid present in said particle.

114. The nucleic acid-lipid particle of any one of claims 104 to 111, wherein the non-cationic lipid comprises a phospholipid.

115. 112. The nucleic acid-lipid particle of any one of claims 104-111, wherein said non-cationic lipid comprises a mixture of phospholipids and cholesterol or a derivative thereof.

116. 116. The nucleic acid-lipid particle of claim 115, wherein said phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or a mixture thereof.

117. The nucleic acid-lipid particle of claim 115, wherein said phospholipid constitutes about 4 mol% to about 10 mol% of the total lipid present in said particle and said cholesterol constitutes about 30 mol% to about 40 mol% of the total lipid present in said particle.

118. The nucleic acid-lipid particle of claim 115, wherein said phospholipid comprises from about 10 mol% to about 30 mol% of the total lipid present in said particle and said cholesterol comprises from about 10 mol% to about 30 mol% of the total lipid present in said particle.

119. 119. The nucleic acid-lipid particle of any one of claims 104-118, wherein the conjugated lipid comprises a polyethylene glycol (PEG)-lipid conjugate.

120. 120. The nucleic acid-lipid particle of claim 119, wherein said PEG-lipid conjugate comprises a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, or a mixture thereof.

121. 121. The nucleic acid-lipid particle of claim 120, wherein said PEG-DAA conjugate comprises a PEG-dimyristyloxypropyl (PEG-DMA) conjugate, a PEG-distearyloxypropyl (PEG-DSA) conjugate, or a mixture thereof.

122. The nucleic acid-lipid particle of claim 120, wherein said PEG-DAA conjugate comprises a PEG-dimyristyloxypropyl (PEG-DMA) conjugate.

123. The polyethylene glycol (PEG)-lipid conjugate has the formula: A-B-C or a salt thereof, wherein A is (C １ -C ６ ) alkyl, (C ３ -C ８ ) cycloalkyl, (C ３ -C ８ ) Cycloalkyl(C １ -C ６ ) alkyl, (C １ -C ６ ) alkoxy, (C ２ -C ６ ) alkenyl, (C ２ -C ６ ) alkynyl, (C １ -C ６ ) alkanoyl, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ ) alkylthio, or (C ２ -C ６ ) alkanoyloxy, where any (C １ -C ６ ) alkyl, (C ３ -C ８ ) cycloalkyl, (C ３ -C ８ ) Cycloalkyl(C １ -C ６ ) alkyl, (C １ -C ６ ) alkoxy, (C ２ -C ６ ) alkenyl, (C ２ -C ６ ) alkynyl, (C １ -C ６ ) alkanoyl, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ ) alkylthio, and (C ２ -C ６ ) alkanoyloxy is substituted with one or more anion precursor groups, and wherein any (C １ -C ６ ) alkyl, (C ３ -C ８ ) cycloalkyl, (C ３ -C ８ ) Cycloalkyl(C １ -C ６ ) alkyl, (C １ -C ６ ) alkoxy, (C ２ -C ６ ) alkenyl, (C ２ -C ６ ) alkynyl, (C １ -C ６ ) alkanoyl, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ ) alkylthio, and (C ２ -C ６ ) alkanoyloxy is optionally selected from halo, hydroxyl, (C １ -C ３ ) alkoxy, (C １ -C ６ ) alkanoyl, (C １ -C ３ ) alkoxycarbonyl, (C １ -C ３ ) alkylthio, or (C ２ -C ３ ) alkanoyloxy; B is a polyethylene glycol chain having a molecular weight of about 550 daltons to about 10,000 daltons; C is -L-R ａ and L is a direct bond, -C(O)O-, or -C(O)NR ｂ --, --NR ｂ -, -C(O)-, -NR ｂ C(O)O-, -NR ｂ C(O)NR ｂ -, -S-S-, -O-, -(O)CCH ２ CH ２ C(O)-, and -NHC(O)CH ２ CH ２ C(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 are substituted with -O-; Each R ｂ are independently H or (C １ -C ６ ) alkyl.

124. The nucleic acid-lipid particle of any one of claims 104 to 123, wherein the PEG has an average molecular weight of about 2,000 daltons.

125. The complex lipid is 【Chemistry 56】 or a salt thereof.

126. The complex lipid has the following formula: 【Chemistry 57】 The compound includes The nucleic acid-lipid particle of any one of claims 93 to 107, wherein n is 45 to 50.

127. 127. The nucleic acid-lipid particle of any one of claims 104-126, wherein the conjugated lipid comprises from about 1 mol % to about 2 mol % of the total lipid present in the particle.

128. The nucleic acid-lipid particle of any one of claims 104 to 127, 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.

129. 129. The nucleic acid-lipid particle of any one of claims 104-128, wherein the nucleic acid is fully encapsulated within the nucleic acid-lipid particle.

130. 130. The nucleic acid-lipid particle of any one of claims 104-129, wherein the nucleic acid-lipid particle has a lipid:nucleic acid mass ratio of about 5 to about 15.

131. 130. The nucleic acid-lipid particle of any one of claims 104-129, wherein the nucleic acid-lipid particle has a lipid:nucleic acid mass ratio of about 5 to about 30.

132. 132. The nucleic acid-lipid particle of any one of claims 104 to 131, wherein the nucleic acid-lipid particle has a median diameter of from about 40 nm to about 150 nm.

133. The nucleic acid-lipid particle of claim 104, wherein said compound or salt constitutes about 56.5 mol% to about 66.5 mol% of the total lipid present in said particle; said non-cationic lipid comprises cholesterol or a derivative thereof constitutes about 31.5 mol% to about 42.5 mol% of the total lipid present in said particle; and said PEG-lipid conjugate constitutes about 1 mol% to about 2 mol% of the total lipid present in said particle.

134. The nucleic acid-lipid particle of claim 133, wherein the nucleic acid-lipid particle comprises about 61.5 mol % of the compound or salt, about 36.9 mol % of cholesterol or a derivative thereof, and about 1.5 mol % of the PEG-lipid conjugate.

135. The nucleic acid-lipid particle of claim 104, wherein said compound or salt constitutes about 52 mol% to about 62 mol% of the total lipid present in said particle; said non-cationic lipid comprises a mixture of phospholipid and cholesterol or a derivative thereof that constitutes about 36 mol% to about 47 mol% of the total lipid present in said particle; and said PEG-lipid conjugate constitutes about 1 mol% to about 2 mol% of the total lipid present in said particle.

136. The nucleic acid-lipid particle of claim 135, wherein the nucleic acid-lipid particle comprises about 57.1 mol % of a compound or salt, about 7.1 mol % of a phospholipid, about 34.3 mol % of cholesterol or a derivative thereof, and about 1.4 mol % of a PEG-lipid conjugate.

137. The nucleic acid-lipid particle of claim 135, wherein the nucleic acid-lipid particle comprises about 57.1 mol % of the compound or salt, about 20 mol % of a phospholipid, about 20 mol % of cholesterol or a derivative thereof, and about 1.4 mol % of a PEG-lipid conjugate.

138. The nucleic acid-lipid particle of claim 104, wherein said compound or salt constitutes 50 mol% to 65 mol% of the total lipid present in said particle; said non-cationic lipid comprises a mixture of phospholipid and cholesterol or a derivative thereof, said phospholipid constitutes 4 mol% to 10 mol% of the total lipid present in said particle, and said cholesterol or a derivative thereof constitutes 30 mol% to 40 mol% of the total lipid present in said particle; and said complex lipid constitutes 0.5 mol% to 2 mol% of the total lipid present in said particle.

139. The nucleic acid-lipid particle of claim 104, wherein said compound or salt constitutes 50 mol% to 65 mol% of the total lipid present in said particle; said non-cationic lipid comprises a mixture of phospholipid and cholesterol or a derivative thereof, said phospholipid constitutes 3 mol% to 15 mol% of the total lipid present in said particle, and said cholesterol or a derivative thereof constitutes 30 mol% to 40 mol% of the total lipid present in said particle; and said complex lipid constitutes 0.5 mol% to 2 mol% of the total lipid present in said particle.

140. The nucleic acid-lipid particle of claim 104, wherein said compound or salt constitutes 50 mol% to 65 mol% of the total lipid present in said particle; said non-cationic lipid constitutes up to 49.5 mol% of the total lipid present in said particle; said cholesterol or derivative thereof constitutes 30 mol% to 40 mol% of the total lipid present in said particle, and said complex lipid constitutes 0.5 mol% to 2 mol% of the total lipid present in said particle.

141. The nucleic acid-lipid particle of claim 104, wherein said compound or salt constitutes 50 mol% to 85 mol% of the total lipid present in said particle; said non-cationic lipid constitutes 13 mol% to 49.5 mol% of the total lipid present in said particle; and said complex lipid constitutes 0.5 mol% to 2 mol% of the total lipid present in said particle.

142. The nucleic acid-lipid particle of claim 104, wherein said compound or salt constitutes about 30 mol% to about 50 mol% of the total lipid present in said particle; said non-cationic lipid comprises a mixture of phospholipid and cholesterol or a derivative thereof that constitutes about 47 mol% to about 69 mol% of the total lipid present in said particle; and said complex lipid constitutes about 1 mol% to about 3 mol% of the total lipid present in said particle.

143. The non-cationic lipid comprises a mixture of cholesterol and DSPC, and the complex lipid comprises 【Chemistry 58】 where n is selected so that the resulting polymer chain has a molecular weight of about 2000 Daltons; The nucleic acid-lipid particle of claim 104, wherein the conjugated lipid comprises about 2 mol% of the total lipid present in the particle; the DSPC comprises about 10 mol% of the total lipid present in the particle; the cholesterol comprises about 48 mol% of the total lipid present in the particle, and the compound or salt comprises about 40 mol% of the total lipid present in the particle.

144. The non-cationic lipid comprises a mixture of cholesterol and DSPC; and the complex lipid comprises 【Chemistry 59】 where n is selected so that the resulting polymer chain has a molecular weight of about 2000 Daltons; The nucleic acid-lipid particle of claim 104, wherein the conjugated lipid comprises about 2 mol% of the total lipid present in the particle; the DSPC comprises about 10 mol% of the total lipid present in the particle; the cholesterol comprises about 48 mol% of the total lipid present in the particle, and the compound or salt comprises about 40 mol% of the total lipid present in the particle.

145. The non-cationic lipid comprises a mixture of cholesterol and DSPC; the complex lipid comprises 【Chemistry 60】 where n is selected so that the resulting polymer chain has a molecular weight of about 2000 Daltons; The nucleic acid-lipid particle of claim 104, wherein said conjugated lipid comprises about 1.6 mol% of the total lipid present in said particle; said DSPC comprises about 10.9 mol% of the total lipid present in said particle; said cholesterol comprises about 32.8 mol% of the total lipid present in said particle; and said compound or salt comprises about 54.9 mol% of the total lipid present in said particle.

146. 146. The nucleic acid-lipid particle of any one of claims 142-145, wherein the lipid to nucleic acid ratio is about 24.

147. The nucleic acid-lipid particle of any one of claims 104 to 146, comprising two or more compounds or salts of any one of claims 1 to 103.

148. 148. A pharmaceutical composition comprising the nucleic acid-lipid particle of any one of claims 104 to 147 and a pharma- ceutically acceptable carrier.

149. 1. A method for introducing a nucleic acid into a cell, comprising: The method comprising contacting said cell with the nucleic acid-lipid particle of any one of claims 104-147 or the composition of claim 148.

150. 150. The method of claim 149, wherein the cell is in a mammal.

151. A method for in vivo delivery of a nucleic acid, comprising: The method comprises administering to a mammalian subject the nucleic acid-lipid particle of any one of claims 104-147 or the composition of claim 148.

152. 152. The method of claim 151, wherein said administration is selected from the group consisting of oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, and intradermal.

153. The nucleic acid-lipid particle of any one of claims 104 to 147 or the composition of claim 148 for use in in vivo delivery of a nucleic acid to a mammal.

154. 150. Use of the nucleic acid-lipid particle of any one of claims 104 to 147 or the composition of claim 148 for the preparation of a medicament for the in vivo delivery of a nucleic acid to a mammal.

155. 1. A method of treating a disease or disorder in a mammalian subject in need of treatment, comprising: The method comprises administering to said mammalian subject a therapeutically effective amount of the nucleic acid-lipid particle of any one of claims 104-147 or the composition of claim 148.

156. 156. The method of claim 155, wherein the disease or disorder is selected from the group consisting of viral infection, liver disease or disorder, and cancer.

157. 149. The nucleic acid-lipid particle of any one of claims 104-147 or the composition of claim 148 for use in treating a disease or disorder in a mammal.

158. 150. Use of the nucleic acid-lipid particle of any one of claims 104 to 147 or the composition of claim 148 for the preparation of a medicament for treating a disease or disorder in a mammal.

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