Improved lipid nanoparticles for delivery of nucleic acids

Abstract

To provide methods and lipid nanoparticles for delivery of a therapeutic agent to primates for treatment of various indications.SOLUTION: A method for delivering a nucleic acid to a primate in need of treatment is provided which comprises administering a lipid nanoparticle (LNP) to the primate, the LNP comprising: i) a nucleic acid, or a pharmaceutically acceptable salt thereof, encapsulated in the LNP; ii) a cationic lipid; iii) a neutral lipid; iv) a steroid; and v) a polymer-conjugated lipid, where a plurality of the LNPs has a mean particle diameter ranging from 40 nm to 70 nm.SELECTED DRAWING: Figure 13

Description

[Technical Field]

[0001] background Embodiments of the present invention generally relate to lipid nanoparticles (LNPs) with improved properties that are useful for enhancing the intracellular delivery of therapeutic agents, such as nucleic acids (e.g., oligonucleotides, messenger RNA), to primates, including humans. [Background technology]

[0002] 2. Description of Related Art Numerous challenges exist in the delivery of nucleic acids to elicit desired responses within biological systems. Nucleic acid-based therapeutics hold great promise, but to realize this potential, there remains a need for more effective delivery of nucleic acids to appropriate sites within cells or organisms. Therapeutic nucleic acids include, for example, messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immunostimulatory nucleic acids, antagomirs, antimirs, mimetics, supermirs, and aptamers. Some nucleic acids, such as mRNA or plasmids, can be used to induce the expression of specific cellular products, useful, for example, for treating diseases associated with protein or enzyme deficiencies. The therapeutic applications of translatable nucleotide delivery are extremely broad, as constructs can be synthesized to produce any selected protein sequence, whether native to the system or not. The expression product of a nucleic acid can increase existing levels of a protein within a cell or organism, replace a missing or non-functional version of a protein, or introduce a new protein and associated functionality.

[0003] However, the use of oligonucleotides in therapeutic settings currently faces challenges. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has limited ability to enter intracellular compartments where the relevant translation machinery resides. Lipid nanoparticles formed from cationic lipids and oligonucleotides with other lipid components, such as neutral lipids, cholesterol, PEG, and PEGylated lipids, have been used to protect RNA in plasma and promote cellular uptake of oligonucleotides.

[0004] Furthermore, lipid nanoparticle formulations show great promise for enhancing nucleic acid therapy in both in vitro and in vivo animal models, and the performance in rodent models is significantly superior to that observed in non-human primate models in almost all measurements, including toxicity and tolerability, pharmacokinetics, tissue targeting and efficacy.In particular, achieving therapeutically relevant results at tolerable dose levels in primate models remains a significant challenge.Therefore, there remains a need for improved lipid nanoparticles for the delivery of oligonucleotides in primates, so that effective and reproducible therapeutic results can be achieved.Embodiments of the present invention provide these and related advantages. Summary of the Invention

[0005]

[0010] Embodiments of the present invention provide improved lipid nanoparticles (LNPs) and methods of use thereof, for example, for the delivery of nucleic acid therapeutic agents to humans and / or non-human primates. In an exemplary embodiment, a method of delivering a nucleic acid to a primate in need of treatment comprises administering to the primate lipid nanoparticles (LNPs), wherein the LNPs: i) a nucleic acid or a pharmaceutically acceptable salt thereof encapsulated in an LNP; ii) cationic lipids; iii) neutral lipids; iv) steroids; and v) 2.0 to 3.5 mole percent polymer-conjugated lipid based on the total moles in lipid of the LNP A method is disclosed that includes:

[0006] In another embodiment, the present invention provides a method of delivering a nucleic acid to a primate comprising administering to the primate in need thereof lipid nanoparticles (LNPs), wherein said LNPs comprise: i) a nucleic acid or a pharmaceutically acceptable salt thereof encapsulated in an LNP; ii) cationic lipids; iii) neutral lipids; iv) steroids; and v) Polymer-conjugated lipids wherein the plurality of LNPs have an average particle size in the range of 40 nm to 70 nm.

[0007] In a further exemplary embodiment, the invention relates to a method of delivering a nucleic acid to a primate, comprising administering to the primate in need thereof lipid nanoparticles (LNPs), wherein said LNPs comprise: i) a nucleic acid or a pharmaceutically acceptable salt thereof encapsulated in an LNP; ii) cationic lipids; iii) neutral lipids; iv) steroids; and v) a polymer-conjugated lipid having the following structure: [ka] [During the ceremony, P is a polymer; L is a trivalent linker 1 to 15 atoms in length; R' and R'' are each independently saturated alkyl having 8 to 14 carbon atoms, provided that the total number of carbon atoms in both R' and R'' is 27 or less. A method is provided, comprising:

[0008] Further embodiments relate to improved components for lipid nanoparticles and lipid nanoparticles comprising the components and uses thereof. For example, one embodiment relates to a lipid nanoparticle having the following structure: [ka] wherein R′, R″, R′″ and n are as defined herein. or a salt thereof. Disclosed are LNPs comprising the above compounds and methods of using them in a variety of methods, including administering therapeutic nucleic acids to primates.

[0009] These and other aspects of various embodiments will become apparent upon reference to the following description.

[0010] In the figures, identical reference numbers identify similar elements. The sizes and relative positions of elements in the figures are not necessarily drawn to scale, and some of these elements have been arbitrarily enlarged and positioned to improve the legibility of the figures. Furthermore, the particular shapes of the elements as drawn are not intended to convey any information about the actual shape of the particular elements, but have been selected merely for ease of recognition in the figures. [Brief explanation of the drawings]

[0011] [Figure 1-2] 1 shows the relative concentration of expressed luciferase in mouse liver for different embodiments of lipid nanoparticles. [Figure 3-4] Figure 1 shows the relative concentration of expressed luciferase in mouse liver for different lipid nanoparticle embodiments as a function of the amount of PEG-lipid in the LNP. [Figure 5] 1 shows the levels of IgG1 present in non-human primate plasma for different embodiments of lipid nanoparticles. [Figure 6] 10 plots the concentration of amino lipids in non-human primate plasma for different embodiments of lipid nanoparticles. [Figure 7] The concentration of amino lipids in non-human primate liver for different embodiments of lipid nanoparticles is plotted as a function of time. [Figure 8-11]1 shows in situ hybridization images showing the distribution of LNPs in specific liver tissue regions for different embodiments of LNPs. [Figure 12] 1 shows cytokine data for cytokines treated with LNPs of Example 4. [Figure 13] Comparison of plasma IgG1 levels for two different sizes of LNP. [Figure 14] IgG expression in mice for two different LNP sizes is shown. [Figure 15] Cytokine data for two different LNP sizes. [Figure 16] 1 shows in situ hybridization images showing the distribution of LNPs in specific liver tissue regions for LNPs of various sizes. [Figure 17] IgG expression in NHPs for two different LNPs. [Figure 18] IgG expression in mice for two different LNPs. [Figure 19] IgG expression data for LNP10-1 and 10-2 are presented. DETAILED DESCRIPTION OF THE INVENTION

[0012] Detailed Description In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, it will be understood by those skilled in the art that the invention may be practiced without these details.

[0013] In certain embodiments, the present invention provides lipid nanoparticles and methods for in vitro and in vivo delivery of mRNA and / or other oligonucleotides.In some embodiments, these improved lipid nanoparticle compositions are useful for the expression of proteins encoded by mRNA.In other embodiments, these improved lipid nanoparticles are useful for upregulating endogenous protein expression by delivering miRNA inhibitors that target a specific miRNA or a group of miRNAs that regulate a single target mRNA or several mRNAs.In other embodiments, these improved lipid nanoparticles are useful for upregulating endogenous protein expression by delivering smaRNAs that target a gene promoter or a group of gene promoters.In other embodiments, these improved lipid nanoparticles are useful for downregulating (e.g., silencing) the protein level and / or mRNA level of target genes.In some other embodiments, lipid nanoparticles are also useful for delivering mRNA, self-amplifying RNA (saRNA) and plasmids for transgene expression. In yet another embodiment, the lipid nanoparticle composition is useful for inducing pharmacological effects resulting from protein expression, such as increased red blood cell production through the delivery of an appropriate erythropoietin mRNA, or protection against infection through the delivery of an mRNA encoding an appropriate antigen or antibody. In yet another embodiment, the lipid nanoparticles can be used for gene editing applications based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology through the delivery of an mRNA capable of expressing Cas9 in combination with an appropriate single guide RNA (sgRNA). Gene editing approaches can be used, for example, to treat hypercholesterolemia by targeting an appropriate gene target, such as PCSK9, in a mouse model of the disease. The lipid nanoparticles of the present invention can be used for a variety of purposes, including delivering encapsulated or bound (e.g., complexed) therapeutic agents, such as nucleic acids, to cells both in vitro and in vivo.Accordingly, an embodiment of the present invention provides a method of administering a therapeutic agent to a patient, such as a primate, in need of treatment, comprising administering to the patient a lipid nanoparticle described herein.

[0014] As described herein, lipid nanoparticle embodiments of the present invention are particularly useful for delivering nucleic acids, including, for example, mRNA, guide RNA, circular RNA, antisense oligonucleotides, plasmid DNA, closed-loop DNA (ceDNA), circular RNA, microRNA (miRNA), miRNA inhibitors (antagomirs / antimirs), messenger RNA interfering complementary RNA (micRNA), self-amplifying RNA (saRNA), small activating RNA (smaRNA), DNA, multivalent RNA, Dicer substrate RNA, complementary DNA (cDNA), peptide nucleic acid (PNA), and the like. Thus, lipid nanoparticles of the present invention can be used to induce the expression of desired proteins both in vitro and in vivo by contacting cells with the lipid nanoparticles. The expressed proteins can have biological effects, such as eliciting an immune response. Alternatively, lipid nanoparticles and compositions of the present invention can be used to reduce the expression of target genes and proteins both in vitro and in vivo by contacting cells with the lipid nanoparticles. The lipid nanoparticles and compositions of the present embodiments may also be used to co-deliver different nucleic acids (e.g., mRNA and plasmid DNA), either separately or in combination, such as may be useful to provide effects requiring co-localization of different nucleic acids (e.g., mRNA encoding an appropriate gene-modifying enzyme with associated guide RNA sequences, if applicable, and optionally one or more DNA segments for integration into the host genome).

[0015] Nucleic acids for use in embodiments of the present invention can be prepared according to the techniques described herein. For mRNA, the primary method of preparation is, but is not limited to, enzymatic synthesis (also called in vitro transcription), which currently represents the most efficient method for generating long-sequence-specific mRNA. In vitro transcription describes the process of template-directed synthesis of RNA molecules from an engineered DNA template composed of an upstream bacteriophage promoter sequence (e.g., including but not limited to, those from T7, T3, and SP6 coliphages) linked to a downstream sequence encoding a gene of interest. Template DNA can be prepared for in vitro transcription from many sources using suitable techniques known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012).

[0016] RNA transcription occurs in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be carried out using a variety of commercially available kits, including, but not limited to, RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription Kit (Life Technologies), and using commercially available reagents, including RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are known in the art (e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46; Kamakaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2: 11.6: 11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. ed.), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter 5 - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, which are incorporated herein by reference). 101-114; see

[0017] The desired in vitro transcribed mRNA is then purified from undesired components of the transcription or related reaction, including unincorporated rNTPs, protein enzymes, salts, short RNA oligos, etc. Techniques for isolating mRNA transcripts are known in the art. Known methods include phenol / chloroform extraction or precipitation with any alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Further, non-limiting examples of purification methods that can be used include size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography, and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA synthesis Methods v. 941 Conn GL (ed.), New York, NY Humana Press, 2012). Purification can be performed using a variety of commercially available kits, including, but not limited to, the SV Total Isolation System (Promega) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0018] Furthermore, while reverse transcription can produce large amounts of mRNA, the product may contain many aberrant RNA impurities associated with undesired polymerase activity that may need to be removed from full-length mRNA preparations. These include short RNAs resulting from incomplete transcription initiation and RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and double-stranded RNA (dsRNA) generated by self-complementary 3' extension. It has been shown that these dsRNA-structured contaminants can result in unwanted immunostimulatory activity through interactions with various innate immune sensors within eukaryotic cells that recognize specific nucleic acid structures and function to induce potent immune responses. This, in turn, can dramatically reduce mRNA translation when protein synthesis is reduced during the cellular innate immune response. Therefore, additional techniques have been developed to remove these dsRNA contaminants, including but not limited to scalable HPLC purification, and are known in the art (e.g., Kariko, K., Muramatsu, H., Ludwig, J. and Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (ed. Rabinovich, PH), 2013). Purified mRNA has been reported to be translated at much greater levels, especially in primary cells and in vivo.

[0019] Various modifications have been described in the art that can be used to alter specific properties of in vitro transcribed mRNA and improve its usefulness. These include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5' end of the mature molecule, which plays an important role in mediating the binding of mRNA cap-binding protein (CBP), which in turn contributes to enhancing intracellular RNA stability and the efficiency of mRNA translation. Therefore, the highest level of protein expression is achieved using capped mRNA transcripts. The 5'-cap contains a 5'-5'-triphosphate bond between the 5'-most nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Further modifications include methylation of the 5'-most and penultimate nucleotides on the 2'-hydroxyl group.

[0020] Several different cap structures can be used to generate 5'-caps for in vitro transcribed synthetic mRNAs. 5'-capping of synthetic mRNAs can be performed by co-transcription with a chemical cap analog (i.e., capping during in vitro transcription). For example, CleanCap® technology provides highly efficient capping (90%+) in co-transcription reactions using commercially available reagents along with an AG initiator to provide a natural Cap 1 structure with a 2'-O-methyl group and an N7 methyl on separate guanine components. As another example, the anti-reverse cap analog (ARCA) cap contains a 5'-5'-triphosphate guanine-guanine linkage in which one guanine contains an N7 methyl group and a 3'-O-methyl group. However, up to 20% of transcripts remain uncapped during this co-transcription process, and synthetic cap analogs are not identical to the 5'-cap structure of authentic cellular mRNAs, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped after transcription. These can produce more authentic 5'-cap structures that more closely mimic endogenous 5'-caps structurally or functionally, with enhanced cap-binding protein binding, extended half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5' decapping. Many synthetic 5'-cap analogs have been developed to enhance mRNA stability and translatability and are known in the art.(e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, RE, Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH ed.), 2013).

[0021] At the 3'-end, a long chain of adenine nucleotides (poly-A tail) is usually added to mRNA molecules during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved, freeing the 3' hydroxyl at which poly-A polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. The poly(A) tail has been widely shown to enhance both the translation efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly(A), poly(A) binding protein and the regulation of mRNA stability, Trends Bio Sci, v. 14, 373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M. and Regnier, P., 2002, The poly(A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v. 111, 611-613).

[0022] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of techniques, including, but not limited to, cloning a poly(T) region into a DNA template or post-transcriptional addition using poly(A) polymerase. The former allows for in vitro transcription of mRNAs with poly(A) tails of defined lengths, depending on the size of the poly(T) region, but requires further manipulation of the template. The latter involves enzymatically adding a poly(A) tail to in vitro transcribed mRNA using poly(A) polymerase, which catalyzes the incorporation of adenine residues into the 3' end of the RNA, without further manipulation of the DNA template, but results in mRNAs with poly(A) tails of heterogeneous lengths. 5'-capping and 3'-poly(A) tailing can be performed using a variety of commercially available kits, including, but not limited to, Poly(A) Polymerase Tailing Kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra Kit and Poly(A) Tailing Kit (Life Technologies), and using commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

[0023] In addition to 5' cap and 3' polyadenylation, other modifications of in vitro transcripts have been reported to provide benefits related to translation efficiency and stability.It is known in the art that pathogen DNA and RNA can be recognized by various sensors in eukaryotic cells, and can trigger a strong natural immune response.Since most naturally occurring nucleic acids contain modified nucleosides, it has been shown that the ability to distinguish between pathogen and self-DNA and RNA is at least partly based on structure and nucleoside modification.In contrast, in vitro synthesized RNA lacks these modifications, and therefore becomes immunostimulatory, which can further inhibit effective mRNA translation as shown above.Introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thereby reducing this undesirable immunostimulatory activity and enhancing translational capacity (e.g., Kariko, K. And Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: Implication for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969(Rabinovich, PH Ed), 2013); Kariko, K., Muramatsu, H., Welsh, FA, Ludwig, J., Kato, H., Akira, S., (See Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833-1840.) Modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared, monitored, and utilized using common methods and procedures known in the art.A wide variety of nucleoside modifications are available that can be incorporated into in vitro transcribed mRNA, either alone or in combination with other modified nucleosides (see, e.g., U.S. Publication No. 2012 / 0251618). In vitro synthesis of nucleoside-modified mRNA has been reported to reduce its ability to activate immune sensors while simultaneously enhancing its translational capacity.

[0024] Other components of mRNA that can be modified to provide benefits in terms of translatability and stability include the 5' and 3' untranslated regions (UTRs). Optimizing both or independently of the UTRs (preferred 5' and 3' UTRs can be obtained from cellular or viral RNA) has been shown to increase mRNA stability and translation efficiency of in vitro transcribed mRNA (see, for example, Pardi, N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PH Ed), 2013).

[0025] In addition to mRNA, other nucleic acid payloads can be used in embodiments of the present invention. For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis of longer precursors, enzymatic or chemical cleavage, and in vitro transcription as described above. Methods for synthesizing DNA and RNA nucleotides are widely used and known in the art (see, for example, Gait, MJ (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005, both of which are incorporated herein by reference).

[0026] For plasmid DNA, preparation for use in embodiments of the present invention typically utilizes, but is not limited to, in vitro propagation and isolation of the plasmid DNA in liquid cultures of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows bacteria containing the plasmid of interest to selectively grow in cultures containing the antibiotic. Methods for isolating plasmid DNA are widely used and known in the art (see, e.g., Heilig, J., Elbing, KL and Brent, R (2001) Large-Scale Preparation of Plasmid DNA. Current Protocols in Molecular Biology. 41: II: 1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstroem, S., Bjoernestedt, R. and Schmidt, SR (2008) Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol. Bioeng., 99: 557-566; and U.S. Pat. No. 6, 197, 553 B1). Plasmid isolation can be performed using a variety of commercially available kits, including but not limited to Plasmid Plus (Qiagen), GenJET Plasmid MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) kits, as well as using commercially available reagents.

[0027] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0028] Unless otherwise required by context, throughout this specification and claims, the word "comprise" and variations thereof, such as "comprises" and "comprising," are to be construed in an open, inclusive sense, i.e., "including but not limited to."

[0029] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with this embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0031] The phrase "induce the expression of a desired protein" refers to the ability of a nucleic acid to increase the expression of a desired protein.To test the level of protein expression, a test sample (e.g., a cell sample in culture that expresses a desired protein) or a test mammal (e.g., a mammal, e.g., a human or an animal model, e.g., a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) is contacted with a nucleic acid (e.g., the nucleic acid combined with the lipid of the present invention).The expression of the desired protein in the test sample or test animal is compared with the expression of the desired protein in a control sample (e.g., a cell sample in culture that expresses a desired protein) or a control mammal (e.g., a mammal, e.g., a human or an animal model, e.g., a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) that has not been contacted with or administered with a nucleic acid.When the desired protein is present in the control sample or control mammal, the expression of the desired protein in the control sample or control mammal can be assigned a value of 1.0. In certain embodiments, induction of expression of a desired protein is achieved when the ratio of desired protein expression in a test sample or test mammal to the level of desired protein expression in a control sample or control mammal is greater than 1, e.g., about 1.1, 1.5, 2.0, 5.0, or 10.0. If the desired protein is not present in the control sample or control mammal, induction of expression of a desired protein is achieved when any measurable level of the desired protein is detected in the test sample or test mammal. Those skilled in the art will recognize appropriate assays for determining protein expression levels in a sample, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function and phenotypic assays, or assays based on reporter proteins capable of producing fluorescence or luminescence under appropriate conditions.

[0032] The phrase " inhibit the expression of target gene " refers to the ability of nucleic acid to silence, reduce or inhibit the expression of target gene.To test the degree of gene silencing, test sample (for example, a cell sample in culture that expresses target gene) or test mammal (for example, a mammal, for example, a human or animal model, for example, a rodent (for example, a mouse) or non-human primate (for example, a monkey) model) is contacted with the nucleic acid that silences, reduces or inhibits the expression of target gene (for example, the nucleic acid combined with lipid of the present invention).The expression of target gene in test sample or test animal is compared with the expression of target gene in control sample (for example, a cell sample in culture that expresses target gene) or control mammal (for example, a mammal, for example, a human or animal model, for example, a rodent (for example, a mouse) or non-human primate (for example, a monkey) model) that is not contacted with nucleic acid or not administered with nucleic acid.The expression of target gene in control sample or control mammal can be assigned a value of 100%. In certain embodiments, silencing, inhibition or reduction of expression of a target gene is achieved when the expression level of the target gene in a test sample or test mammal is about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or about 0% of the level of target gene expression in a control sample or control mammal. In other words, the nucleic acid is capable of silencing, reducing or inhibiting expression of the target gene in a test sample or test mammal by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% relative to the expression level of the target gene in a control sample or control mammal not contacted with or administered the nucleic acid.Suitable assays for determining the level of target gene expression include, but are not limited to, testing protein or mRNA levels using techniques known to those of skill in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzymatic function and phenotypic assays known to those of skill in the art.

[0033] An "effective amount" or "therapeutically effective amount" of a therapeutic agent, such as an active agent or therapeutic nucleic acid, is an amount sufficient to produce a desired effect, e.g., increased or inhibited expression of a target sequence compared to normal expression levels detected in the absence of the nucleic acid. Increased expression of a target sequence is achieved when any measurable level is detected in the case of expression product that is not present in the absence of the nucleic acid. When the expression product is present at some level prior to contact with the nucleic acid, increased expression is achieved when the fold increase over that obtained with a nucleic acid such as mRNA compared to a control is about 1.05, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.75, about 2, about 2.5, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 250, about 500, about 750, about 1000, about 5000, about 10000 or more. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as an antisense oligonucleotide is about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 0% compared to a control. Suitable assays for measuring expression of a target gene or target sequence include, for example, testing protein or mRNA levels using techniques known to those skilled in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art.

[0034] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single- or double-stranded form, including DNA, RNA, and mixtures thereof. DNA can be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA can be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), self-amplifying RNA (saRNA), small activator RNA, antisense RNA, miRNA, micRNA, polyvalent RNA, Dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include synthetic, natural, and unnatural nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that 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-methyl ribonucleotides, 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 specified, a specific nucleic acid sequence also implicitly encompasses its traditionally modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via phosphate groups."Base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogues, and synthetic derivatives of purines and pyrimidines, which include modifications that place new reactive groups such as, for example, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0035] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that includes a portion or entire length of a sequence encoding a polypeptide or precursor polypeptide or that provides for the regulation of gene expression. "Gene" can refer to both coding and non-coding (non-protein-encoding) nucleic acid sequences. For example, non-coding "genes" can be transcribed into functional RNA products, including regulatory RNAs, transfer RNAs (tRNAs), microRNAs (miRNAs), and ribosomal RNAs (rRNAs).

[0036] As used herein, "gene product" refers to gene products such as RNA transcripts, including coding and non-coding variants or polypeptides.

[0037] The term "lipid" refers to a group of organic compounds generally characterized by being poorly soluble in water but soluble in many organic solvents, including, but not limited to, fatty acid esters. They are usually divided into at least three classes: (1) "simple lipids," including fats, oils, and waxes; (2) "compound lipids," including phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0038] "Steroids" are compounds with the following carbon skeleton: [ka] Non-limiting examples of steroids include cholesterol, and the like.

[0039] "Cationic lipid" refers to a lipid that can be positively charged. Typical cationic lipids contain one or more amine groups with a positive charge. Preferred cationic lipids are ionizable, so that they can exist in a positively charged or neutral form depending on pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under various pH conditions. This charge state is important for plasma protein absorption, blood clearance and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17(1998)) and the non-bilayer structure that is important for intracellular delivery of nucleic acids (Hafez, IM, et al., Gene Ther 8:1188-1196(2001)).

[0040] "Anionic lipid" refers to a lipid that can be negatively charged. Typical anionic lipids include, for example, one or more phosphate groups that are negatively charged at physiological pH. In some embodiments, anionic lipids do not include serine moieties, including phosphatidylserine lipids.

[0041] "Phosphatidylglycerol lipid" refers to a lipid having a structure generally comprising a glycerol 3-phosphate backbone to which saturated or unsaturated fatty acids are attached via ester bonds. A typical phosphatidylglycerol lipid has the following structure: [ka] wherein R1 and R2 are each independently a branched or straight-chain saturated or unsaturated carbon chain (e.g., alkyl, alkenyl, alkynyl).

[0042] The term "polymer-conjugated lipid" refers to a molecule that contains both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like. The term "PEGylated lipid" is used interchangeably with "PEGylated lipid."

[0043] The term "neutral lipid" refers to any of a number of lipids that exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), steroids such as ceramides, sterols, and their derivatives. Neutral lipids can be synthetic or naturally occurring. Neutral lipids include lipids sometimes referred to as "non-cationic" lipids.

[0044] The term "charged lipid" refers to any of a number of lipid species that exist in positively or negatively charged form regardless of pH within a useful physiological range, e.g., from about pH 3 to about pH 9. Charged lipids can be synthetic or naturally occurring. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (e.g., DC-Chol).

[0045] The term "lipid nanoparticle" refers to a particle having at least one dimension on the order of nanometers (e.g., 1 to 1,000 nm) that comprises one or more specific lipids. In some embodiments, the lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a desired target site (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticles of the present invention comprise a nucleic acid. Such lipid nanoparticles typically comprise a cationic lipid selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids, and one or more excipients. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, can be encapsulated in the lipid portion of the lipid nanoparticle or in the aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by mechanisms of the host organism or cells, such as a harmful immune response.

[0046] In various embodiments, the lipid nanoparticles are about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, about 40 nm to about 50 nm, about 40 nm to about 60 nm, about 40 nm to about 70 nm, about 40 nm to about 80 nm, about 45 nm to about 50 nm, about 45 nm to about 55 nm, about 45 nm to about The lipid nanoparticles have an average diameter of 60 nm, about 45 nm to about 65 nm, about 45 nm to about 70 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 70 nm, about 55 nm to about 65 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, when present in the lipid nanoparticles, the nucleic acid is resistant to degradation by nucleases in aqueous solution.Nucleic acids and methods for their preparation are described in, for example, U.S. Patent Nos. 8,569,256, 5,965,542 and U.S. Patent Publication Nos. 2016 / 0199485, 2016 / 0009637, 2015 / 0273068, 2015 / 0265708, 2015 / 0203446, 2015 / 0005363, 2014 / 0308304, 2014 / 0200257, 2013 / 086373, 2014 / 0308304, 2014 / 0200257, 2013 / 086373, No. 2013 / 0338210, No. 2013 / 0323269, No. 2013 / 0245107, No. 2013 / 0195920 No., No. 2013 / 0123338, No. 2013 / 0022649, No. 2013 / 0017223, No. 2012 / 029583 No. 2, No. 2012 / 0183581, No. 2012 / 0172411, No. 2012 / 0027803, No. 2012 / 00581 No. 88, No. 2011 / 0311583, No. 2011 / 0311582, No. 2011 / 0262527, No. 2011 / 0216 No. 622, No. 2011 / 0117125, No. 2011 / 0091525, No. 2011 / 0076335, No. 2011 / 00 No. 60032, No. 2010 / 0130588, No. 2007 / 0042031, No. 2006 / 0240093, No. 2006 / 0 No. 083780, No. 2006 / 0008910, No. 2005 / 0175682, No. 2005 / 017054, No. 2005 / 0 No. 118253, No. 2005 / 0064595, No. 2004 / 0142025, No. 2007 / 0042031, No. 1999 / 009076 and PCT Publication Nos. WO 99 / 39741, WO 2017 / 117528, WO 2017 / 004143, WO 2017 / 075531, WO 2015 / 199952, WO 2014 / 008334, WO 2013 / 086373, WO 2013 / 086322, WO 2013 / 016058, WO 2013 / 086373, WO 2011 / 141705, and WO 2001 / 07548. LNPs are prepared according to the methods disclosed herein.

[0047] Other exemplary lipids and their preparation are known in the art, e.g., U.S. Patent Application Publication No. US 2012 / 0276209; Semple et al., 2010, Nat Biotechnol., 28(2):172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther Nucleic Acids, 1: e37; Jayaraman et al. al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, e139; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each of which is incorporated by reference in its entirety. Lipids and their preparation can be found in U.S. Publication Nos. 2015 / 0376115 and 2016 / 0376224, both of which are incorporated by reference herein.

[0048] As used herein, " encapsulated lipid " refers to lipid nanoparticles that provide active or therapeutic agents, such as nucleic acids (e.g., mRNA), by completely encapsulating, partially encapsulating, or both.In some embodiments, nucleic acids (e.g., mRNA) are completely encapsulated in lipid nanoparticles.

[0049] As used herein, the term "aqueous solution" refers to a composition that includes water.

[0050] "Serum stable" in relation to nucleic acid-lipid nanoparticles means that the nucleotides are not significantly degraded after exposure to serum or nuclease assays that can significantly degrade free DNA or RNA.Suitable assays include, for example, standard serum assays, DNase assays, or RNase assays.

[0051] As used herein, "systemic delivery" refers to the delivery of therapeutic products that can result in widespread exposure to active agents in the body of an organism.Some administration techniques can result in systemic delivery of certain drugs, while others cannot.Systemic delivery means that a useful amount, preferably a therapeutic amount, of a drug is exposed to most of the body.Systemic delivery of lipid nanoparticles can be by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous and intraperitoneal delivery.In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0052] As used herein, "local delivery" refers to the direct delivery of active agents to target sites within an organism.For example, agents can be delivered locally by direct injection into disease sites, such as tumors, other target sites, such as inflammation sites, or target organs, such as the liver, heart, pancreas, kidney, etc.Local delivery can also include local application or local injection techniques, such as intramuscular, subcutaneous, or intradermal injection.Local delivery does not interfere with systemic pharmacological effects.

[0053] "Amino acid" refers to natural amino acids and non-natural amino acids. Amino acid lipids can be made from genetically encoded amino acids, natural non-genetically encoded amino acids, or synthetic amino acids. Examples of amino acids include Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. Examples of amino acids also include azetidine, 2-aminooctadecanoic acid, 2-aminoadipic acid, 3-aminoadipic acid, 2,3-diaminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 2,3-diaminobutyric acid, 2,4-diaminobutyric acid, 2-aminoisobutyric acid, 4-aminoisobutyric acid, 2-aminopimelic acid, 2,2'-diaminopimelic acid, 6-aminohexanoic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, desmosine, ornithine, citrulline, N-methylisoleucine, norleucine, tert-leucine, phenylglycine, t-butylglycine, N-methylglycine, sarcosine, N-ethylglycine, cyclohexylglycine, 4-oxo-cyclohexylglycine, N-ethylasparagine, cyclohexylalanine, t-butylalanine, naphthylalanine, pyridyl "Amino acids" include alanine, 3-chloroalanine, 3-benzothienylalanine, 4-halophenylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 2-thienylalanine, methionine, methionine sulfoxide, homoarginine, norarginine, nor-norarginine, N-acetyllysine, 4-aminophenylalanine, N-methylvaline, homocysteine, homoserine, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, 6-N-methyllysine, norvaline, O-allylserine, O-allylthreonine, α-aminohexanoic acid, α-aminovaleric acid, pyroglutamic acid, and derivatives thereof. "Amino acids" include α- and β-amino acids.Examples of amino acid residues can be found in Fasman, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc. (1989).

[0054] "Alkyl" refers to an alkyl group that is saturated or unsaturated (i.e., contains one or more double bonds (alkenyl) and / or triple bonds (alkynyl)), e.g., 1 to 24 carbon atoms (C1-C 24 alkyl), 4 to 12 carbon atoms (C4-C 20 alkyl), 6 to 16 carbon atoms (C6-C 16 alkyl), 6 to 9 carbon atoms (C6-C9 alkyl), 1 to 15 carbon atoms (C1-C 15 alkyl), 1 to 12 carbon atoms (C1-C 12 "Ci-C alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms having from 1 to 8 carbon atoms (Ci-C alkyl), or from 1 to 6 carbon atoms (Ci-C alkyl), attached to the rest of the molecule by a single bond. Unless otherwise specified in the specification, an alkyl group may be optionally substituted.

[0055] An "alkylene" or "alkylene chain" is an alkylene group that is saturated or unsaturated (i.e., contains one or more double bonds (alkenylene) and / or triple bonds (alkynylene)), e.g., 1 to 24 carbon atoms (C-C 24 Alkylene, 1 to 15 carbon atoms (C4-C 20 Alkylene, 1 to 15 carbon atoms (C1-C 15 Alkylene, 1 to 12 carbon atoms (C1-C 12"C-C alkylene" refers to a straight or branched divalent hydrocarbon chain having 1 to 8 carbon atoms (C-C alkylene), 1 to 6 carbon atoms (C-C alkylene), 2 to 4 carbon atoms (C-C alkylene), or 1 or 2 carbon atoms (C-C alkylene), connecting a radical to the rest of the molecule consisting solely of carbon and hydrogen, e.g., methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is attached to the rest of the molecule by a single or double bond and to the radical group by a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons in the chain. Unless stated otherwise in the specification, an alkylene chain can be optionally substituted.

[0056] The term "alkenyl" refers to an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyl includes both cis and trans isomers. Representative straight-chain and branched alkenyls include, but are not limited to, ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

[0057] "Alkoxy" refers to an alkyl, cycloalkyl, alkenyl, or alkynyl group covalently linked to an oxygen atom.

[0058] "Alkanoyloxy" refers to an -OC(=O)-alkyl group.

[0059] "Alkylamino" refers to the group -NRR', where R and R' are each hydrogen or alkyl, and at least one of R and R' is alkyl. Alkylamino refers to groups such as piperidino, where R and R' form a ring. The term "alkylaminoalkyl" refers to -alkyl-NRR'.

[0060] The term "alkynyl" refers to any alkyl or alkenyl as defined above containing at least one triple bond between adjacent carbon atoms. Representative straight-chain and branched alkynyls include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

[0061] The terms "acyl," "carbonyl," and "alkanoyl" refer to any alkyl, alkenyl, or alkynyl, as defined below, in which the carbon at the point of attachment is substituted with an oxo group. The following are non-limiting examples of acyl, carbonyl, or alkanoyl groups: -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl.

[0062] "Aryl" refers to any stable monocyclic, bicyclic, or polycyclic carbocyclic ring system having 4 to 12 atoms in each ring, with at least one ring being aromatic. Some examples of aryl include phenyl, naphthyl, tetrahydronaphthyl, indanyl, and biphenyl. When an aryl substituent is bicyclic and one ring is non-aromatic, it is understood that the bond is to the aromatic ring. Aryl can be substituted or unsubstituted.

[0063] "Carboxyl" refers to a functional group of formula -C(=O)OH.

[0064] "Cyano" refers to a functional group of formula -CN.

[0065] "Cycloalkyl" or "carbocyclic ring" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, and which is saturated or unsaturated and has 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, attached to the remainder of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise specified in the specification, cycloalkyl groups may be optionally substituted.

[0066] A "cycloalkylene" is a divalent cycloalkyl group. Unless stated otherwise in the specification, a cycloalkylene group may be optionally substituted.

[0067] The term "diacylglycerol" or "DAG" includes compounds having two fatty acyl chains of 2 to 30 carbons, both independently attached to the 1- and 2-positions of glycerol by ester bonds. The acyl groups can be saturated or have different degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C12), myristoyl (C14), palmitoyl (C16), stearoyl (C18), and icosoyl (C20). In preferred embodiments, the fatty acyl chains of a compound are the same, i.e., both myristoyl (i.e., dimyristoyl), both stearoyl (i.e., distearoyl), etc.

[0068] The term "heterocycle" or "heterocyclyl" refers to an aromatic or non-aromatic ring system of 5 to 22 atoms in which 1 to 4 ring atoms are heteroatoms selected from oxygen, nitrogen, and sulfur. Thus, a heterocycle can be a heteroaryl or its dihydro or tetrahydro form. Heterocycles include, but are not limited to, pyrrolidine, tetrahydrofuran, thiolane, azetidine, oxetane, thietane, diazetidine, dioxetane, dithietane, piperidine, tetrahydrofuran, pyran, tetrahydropyran, thiacyclohexane, tetrahydrothiophene, pyridine, pyrimidine, and the like.

[0069] "Heteroaryl" refers to any stable monocyclic, bicyclic, or polycyclic carbocyclic ring system having 4 to 12 atoms in each ring, in which at least one ring is aromatic and contains 1 to 4 heteroatoms selected from oxygen, nitrogen, and sulfur. Some examples of heteroaryl include acridinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinolinyl. Heteroaryl includes the N-oxide derivatives of nitrogen-containing heteroaryls.

[0070] The terms "alkylamine" and "dialkylamine" refer to the --NH(alkyl) and --N(alkyl)2 radicals, respectively.

[0071] The term "alkyl phosphate" refers to --O---P(Q')(Q")-O---R, where Q' and Q" are each independently O, S, N(R), optionally substituted alkyl, or alkoxy; and R is optionally substituted alkyl, ω-aminoalkyl, or ω-(substituted)aminoalkyl.

[0072] The term "alkyl phosphorothioate" refers to an alkyl phosphate where at least one of Q' or Q'' is S.

[0073] The term "alkylphosphonate" refers to an alkylphosphate where at least one of Q' or Q'' is alkyl.

[0074] "Hydroxyalkyl" refers to the group ---O-alkyl.

[0075] The term "alkylheterocycle" refers to an alkyl in which at least one methylene is replaced with a heterocycle.

[0076] The term "ω-aminoalkyl" refers to an -alkyl-NH radical, and the term "ω-(substituted)aminoalkyl" refers to an ω-aminoalkyl in which at least one of the H on the N is replaced with an alkyl.

[0077] The term "ω-phosphoalkyl" refers to -alkyl-O---P(Q')(Q'')-O---R, where Q' and Q'' are each independently O or S, and R is an optionally substituted alkyl.

[0078] The term "ω-thiophosphoalkyl" refers to an ω-phosphoalkyl where at least one of Q' or Q'' is S.

[0079] As used herein, the term "substituted" means that at least one hydrogen atom has been replaced with a non-hydrogen atom, for example, but not limited to, a halogen atom such as F, Cl, Br, or I; an oxo group (=O); a hydroxyl group (-OH); a C-C 12 Alkyl group; Cycloalkyl group; -(C=O)OR'; -O(C=O)R'; -C(=O)R'; -OR'; -S(O) x R';-S-SR';-C(=O)SR';-SC(=O)R';-NR'R';-NR'C(=O)R';-C(=O)NR'R';-NR'C(=O)NR'R';-OC(=O)NR'R';-NR'C(=O)OR';-NR'S(O) x NR'R';-NR'S(O) x R'; and -S(O) xNR' means any of the above groups (e.g., alkyl, alkylene, cycloalkyl, or cycloalkylene) substituted by a bond to R', wherein R', in each occurrence, is independently H, C-C 15 alkyl or cycloalkyl, and x is 0, 1, or 2. In some embodiments, the substituent is C-C 12 In another embodiment, the substituent is an alkyl group. In another embodiment, the substituent is a cycloalkyl group. In another embodiment, the substituent is halo, e.g., fluoro. In another embodiment, the substituent is an oxo group. In another embodiment, the substituent is a hydroxyl group. In another embodiment, the substituent is an alkoxy group (-OR'). In another embodiment, the substituent is a carboxyl group. In another embodiment, the substituent is an amine group (-NR'R').

[0080] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described circumstance may or may not occur, and that the description includes cases where said event or circumstance occurs and cases where it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may be substituted or unsubstituted, and that the description includes both substituted alkyl groups and alkyl groups that have no substituents.

[0081] The term "prodrug" refers to a compound, such as a therapeutic agent, that can be converted into a biologically active compound of the present invention under physiological conditions or by solvolysis. Thus, a "prodrug" refers to a pharmaceutically acceptable metabolic precursor of a compound of the present invention. When administered to a subject in need of treatment, a prodrug may be inactive, but is converted in vivo to an active compound of the present invention. Prodrugs are typically rapidly converted in vivo, for example, by hydrolysis in blood, to yield the original compound of the present invention. Prodrug compounds often offer advantages of solubility, tissue compatibility, or delayed release in mammals (see Bundgard, H., Design of Prodrugs (1985), pp. 79, 2124 (Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi, T., et al., ACS Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987).

[0082] The term "prodrug" also includes any covalently bonded carrier, which liberates an active compound of the invention in vivo when the prodrug is administered to a mammalian subject. Prodrugs (e.g., prodrugs of therapeutic agents) can be prepared by modifying functional groups present in the compounds of the invention such that the modifications are cleaved in routine manipulation or in vivo back to the compound of the invention. Prodrugs include compounds in which a hydroxy group, an amino group, or a mercapto group is bonded to any group such that the hydroxy group, the amino group, or the mercapto group, respectively, is cleaved to a free hydroxy group, a free amino group, or a free mercapto group when the prodrug is administered to a mammalian subject. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohols or amide derivatives of amine functional groups in, for example, therapeutic agents of the invention.

[0083] The embodiments of the invention disclosed herein are also meant to encompass all pharmaceutically acceptable lipid nanoparticles and components thereof (e.g., cationic lipids, therapeutic agents, etc.) that are isotopically labeled by replacing one or more atoms with atoms having a different atomic mass or mass number. Examples of isotopes that may be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, e.g., 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I and 125 I, respectively. These radiolabeled LNPs can be useful for determining or assisting in measuring the efficacy of compounds, for example, by characterizing the site or mechanism of action, or binding affinity to a pharmacologically important site of action. Certain isotopically labeled LNPs, for example, LNPs containing radioisotopes, are useful in drug and / or substrate tissue distribution studies. The radioisotope tritium, i.e., 3 H and carbon-14, i.e., 14 C are particularly useful for this purpose, especially in view of their ease of incorporation and facile methods of detection.

[0084] Heavier isotopes, such as deuterium, i.e. 2 Substitution with H may offer certain therapeutic advantages resulting from extremely high metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and may therefore be preferred in some circumstances.

[0085] Positron-emitting isotopes, e.g. 11 C. 18 F, 15 O and 13Substitution at N may be useful in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds used in the present invention may generally be prepared by conventional techniques known to those skilled in the art, or by methods analogous to those described in the examples set forth below, substituting appropriate isotopically labeled reagents for the previously used unlabeled compounds.

[0086] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0087] "Mammal" includes humans and both domestic animals, such as laboratory animals and farm animals (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domestic animals, such as wild animals. "Primate" includes both human and non-human primates.

[0088] "Pharmaceutically acceptable carrier, diluent, or excipient" includes any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the U.S. Food and Drug Administration that is acceptable for use in humans or domestic animals.

[0089] "Pharmaceutically acceptable salts" include both acid and base addition salts.

[0090] "Pharmaceutically acceptable acid addition salts" refers to salts that retain the biological effectiveness and properties of the free bases, which are biologically or otherwise desirable, and include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well as acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfonic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, It is formed using organic acids such as glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0091] "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acids, and are biologically or otherwise desirable. These salts are prepared from the addition of an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0092] "Pharmaceutical composition" refers to a vehicle generally accepted in the art for delivering formulations of LNPs and biologically active compounds of the invention to mammals, e.g., humans. Such a vehicle includes all pharmaceutically acceptable carriers, diluents, or excipients thereof.

[0093] "Effective amount" or "therapeutically effective amount" refers to the amount of the compound of the present invention sufficient for effective treatment of a mammal, preferably a human. The amount of lipid nanoparticles of the present invention that constitutes a "therapeutically effective amount" varies depending on the compound, the condition and its severity, the method of administration and the age of the mammal to be treated, but can be routinely determined by those skilled in the art taking into account their own knowledge and the present invention.

[0094] As used herein, "treating" or "treatment" includes administering to a mammal, preferably a human, a mammal having the disease or condition of interest, and ... (i) preventing the disease or condition from developing in the mammal, particularly where such mammal is susceptible to the condition but has not yet been diagnosed as having it; (ii) inhibiting the disease or condition, i.e., halting its progression; (iii) alleviating the disease or condition, i.e., causing regression of the disease or condition; or (iv) Relieving symptoms resulting from a disease or condition, i.e., alleviating suffering without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably, or the particular disease or condition may not have a known causative agent (and thus the etiology has not yet been elucidated) and therefore is not yet recognized as a disease, but only as an undesirable state or syndrome for which a more or less specific set of symptoms has been identified by clinicians.

[0095] Lipid nanoparticles and methods of use thereof Embodiments described herein relate to methods of using LNPs to deliver therapeutic agents, such as nucleic acids, to primates, such as humans, for the treatment of a variety of diseases treatable with nucleic acids. The present applicants have discovered that the disclosed methods are surprisingly more effective for delivering the same therapeutic agents to primates compared to non-primates, such as mice. For example, some methods involve the use of LNPs with smaller diameters than typical LNPs, e.g., average particle sizes in the range of about 40-70 nm, or, for example, average particle sizes in the range of about 50-70 nm, and such LNPs have unexpectedly improved delivery in primates compared to rodents. Other methods involve the use of LNPs with higher concentrations of PEGylated lipids (e.g., about 2.0-3.5%). Another exemplary method involves delivering LNPs to primates, the LNPs comprising PEGylated lipids having two acyl chains, each independently containing 8-14 carbon atoms, with the total number of carbon atoms in the acyl chains not exceeding 27. The LNPs can be delivered intravenously or by routes of administration known in the art. Further details of these exemplary embodiments and others will be apparent from the details provided herein.

[0096] Thus, in one embodiment, there is provided a method of delivering a nucleic acid to a primate in need thereof, comprising administering to the primate lipid nanoparticles (LNPs), wherein said LNPs comprise: i) a nucleic acid or a pharmaceutically acceptable salt thereof encapsulated in an LNP; ii) cationic lipids; iii) neutral lipids; iv) steroids; and v) 2.0 to 3.5 mole percent polymer-conjugated lipid, based on the total moles in lipid of the LNP A method is provided, comprising:

[0097] The molar percentage of polymer-conjugated lipids is determined based on the total molar percentage of lipids present in the LNP. For this calculation, all lipid components are included, including, for example, cationic lipids, neutral lipids, steroids, and any other lipids, such as anionic or other lipids.

[0098] In certain embodiments, the LNP comprises 2.0 to 3.4 moles of polymer-conjugated lipid. In other embodiments, the LNP comprises 2.1 to 3.5 moles of polymer-conjugated lipid. In further embodiments, the LNP comprises 2.2 to 3.3 mole percent of polymer-conjugated lipid, e.g., 2.3 to 2.8 mole percent of polymer-conjugated lipid. In other embodiments, the LNP comprises 2.1 to 2.5 mole percent of polymer-conjugated lipid. In other different embodiments, the LNP comprises 2.5 to 2.9 mole percent of polymer-conjugated lipid. In other embodiments, the LNP comprises 2.4 to 2.6 mole percent of polymer-conjugated lipid, 2.6 to 2.8 mole percent of polymer-conjugated lipid, 2.4 to 2.5 mole percent of polymer-conjugated lipid, or 2.5 to 2.7 mole percent of polymer-conjugated lipid. In yet different embodiments, the LNP comprises about 2.3 mole percent, about 2.35 mole percent, about 2.4 mole percent, about 2.45 mole percent, about 2.5 mole percent, about 2.55 mole percent, about 2.6 mole percent, about 2.65 mole percent, about 2.7 mole percent, about 2.75 mole percent, or about 2.8 mole percent of the polymer-conjugated lipid.

[0099] Another embodiment is a method of delivering a nucleic acid to a primate comprising administering to the primate in need thereof lipid nanoparticles (LNPs), wherein the LNPs comprise: i) a nucleic acid or a pharmaceutically acceptable salt thereof encapsulated in an LNP; ii) cationic lipids; iii) neutral lipids; iv) steroids; and v) Polymer-conjugated lipids wherein a plurality of said LNPs have an average particle size in the range of 40 nm to 70 nm.

[0100] In certain embodiments, the average particle size is within the range of 45 nm to 70 nm, 50 nm to 70 nm, 55 nm to 65 nm, 50 nm to 60 nm, or 60 nm to 70 nm. In different embodiments, the average particle size is within the range of 45 nm to 50 nm, 50 nm to 55 nm, 55 nm to 60 nm, 60 nm to 65 nm, or 65 nm to 70 nm. In further embodiments, the average particle size is about 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, about 51 nm, about 52 nm, about 53 nm, about 54 nm, about 55 nm, about 56 nm, about 57 nm, about 58 nm, about 59 nm, about 60 nm, about 61 nm, about 62 nm, about 63 nm, about 64 nm, or about 65 nm, about 66 nm, about 67 nm, about 68 nm, about 69 nm, or about 70 nm.

[0101] In any of the above embodiments, the polymer-conjugated lipid has the structure: [ka] [During the ceremony, P is a polymer; L is a trivalent linker 1 to 15 atoms in length; R' and R'' are each independently a saturated alkyl having 8 to 14 carbon atoms.

[0102] In some embodiments, P comprises a polyethylene glycol polymer, e.g., a hydroxyl- or alkoxyl-terminated (PEG-OR) polyethylene glycol polymer. A hydroxyl-terminated polyethylene glycol polymer (PEG-OH) is a polyethylene glycol polymer that terminates with a hydroxyl group, while an alkoxyl-terminated polyethylene glycol polymer (PEG-OR) is a polyethylene glycol polymer that terminates with an alkoxyl group, such as methoxy.

[0103] Any suitable linker can be used for L. In some exemplary embodiments, L comprises an amide, ester, and / or carbamate functional group. For example, in some embodiments, the polymer-conjugated lipid has one of the following structures: [ka] [During the ceremony, n is an integer ranging from 30 to 60; R' and R'' are each independently a saturated alkyl having 8 to 14 carbon atoms; R''' is H or C1-C6 alkyl.

[0104] In other more specific embodiments, the polymer-conjugated lipid has the following structure: [ka] [During the ceremony, n is an integer ranging from 40 to 50; Each R is a saturated alkyl having 8 to 14 carbon atoms, or 8 to 12 carbon atoms, or 8 carbon atoms, or 10 carbon atoms, or 12 carbon atoms. In some embodiments, each R is 8, or each R is 9, or each R is 10, or each R is 11, or each R is 12, or each R is 13, or each R is 14. Embodiments in which each R is not the same are also contemplated, such as one R is 12 and the other R is 13, or one R is 13 and the other R is 14, or one R is 11 and the other R is 12, or one R is 10 and the other R is 11, etc.

[0105] In another different embodiment, the polymer-conjugated lipid has the structure: [ka] [During the ceremony, R 3 HA-OR Oand; R O is hydrogen or alkyl; r is an integer ranging from 30 to 60 (inclusive); R 5 is C 10-20 alkyl]

[0106] For example, in certain embodiments: R 3 is OH or OCH3; R 5 is C 18 , C 19 or C 20 and; r is [ka] is selected to have an average molecular weight of 1,800 Da to 2,200 Da.

[0107] In yet another embodiment, there is provided a method of delivering a nucleic acid to a primate comprising administering to the primate in need thereof lipid nanoparticles (LNPs), wherein said LNPs comprise: i) a nucleic acid or a pharmaceutically acceptable salt thereof encapsulated in an LNP; ii) cationic lipids; iii) neutral lipids; iv) steroids; and v) a polymer-conjugated lipid having the structure: [ka] [During the ceremony, P is a polymer; L is a trivalent linker 1 to 15 atoms in length; R' and R'' are each independently a saturated alkyl group having 8 to 14 carbon atoms; provided that the total number of carbon atoms in both R' and R'' is 27 or less. A method is provided, comprising:

[0108] In certain of the foregoing embodiments, P comprises a polyethylene glycol polymer, eg, a hydroxyl- or alkoxyl-terminated polyethylene glycol polymer.

[0109] In other embodiments, L comprises an amide, ester and / or carbamate functional group, for example, in some embodiments, the polymer-conjugated lipid has one of the following structures: [ka] [During the ceremony, R''' is H or C1-C6 alkyl; n is an integer ranging from 30 to 60.

[0110] In a more specific embodiment, the polymer-conjugated lipid has the following structure: [ka] (wherein n is an integer ranging from 40 to 50)

[0111] In certain of the above embodiments, the total number of carbon atoms in R' and R'' is in the range of 16 to 25, 16 to 24, 17 to 24, or 18 to 24. For example, in some embodiments: a) R' and R'' are each saturated alkyl having 8 carbon atoms; b) R' and R'' are each saturated alkyl having 9 carbon atoms; c) R' and R'' are each saturated alkyl having 10 carbon atoms; d) R' and R'' are each saturated alkyl having 11 carbon atoms; e) R' and R'' are each saturated alkyl having 12 carbon atoms; or f) R' and R'' are each saturated alkyl having 13 carbon atoms.

[0112] Asymmetric polymer-conjugated lipids in which R' and R'' are not identical are also included in various embodiments, such as R' is 12 and R'' is 13, or R' is 13 and R'' is 14, or R' is 11 and R'' is 12, or R' is 10 and R'' is 11, etc.

[0113] In some embodiments, the lipid nanoparticles comprise a cationic lipid, a PEGylated lipid, a sterol, and a neutral lipid. In some embodiments, the lipid nanoparticles comprise a molar ratio of about 20-60% cationic lipid: about 5-25% neutral lipid: about 25-55% sterol; and about 0.1-15% PEGylated lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid. In some embodiments, the neutral lipid is a phospholipid. In some embodiments, the sterol is cholesterol. In some embodiments, the cationic lipid is 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (DLin-KC2-DMA). 3 19). In some embodiments, the lipid nanoparticles have a polydispersity of less than 0.4. In some embodiments, the lipid nanoparticles have a net neutral charge at neutral pH. In some embodiments, the lipid nanoparticles have a mean diameter of 40-200 nm.

[0114] Lipid nanoparticles comprise one or more lipid species, including, but not limited to, cationic / ionizable lipids, neutral lipids, structural lipids, phospholipids, and helper lipids. Any of these lipids may be conjugated with polyethylene glycol (PEG), thereby referred to as PEGylated or PEG-modified lipids.

[0115] Formulation of lipid nanoparticles (LNPs) can be achieved by methods known in the art and / or as described in U.S. Patent Publication No. 2012 / 0178702, which is incorporated herein by reference in its entirety.

[0116] Lipid nanoparticle formulations can be influenced by, but are not limited to, the selection of cationic lipid components, the saturation of cationic lipids, the selection of neutral lipid components, the saturation of neutral lipids, the selection of structural lipid components, the nature of PEGylation, and the ratio of biophysical parameters such as all components and size.In a specific, non-limiting example, LNPs comprise four basic components: (1) cationic lipids; (2) neutral lipids (e.g., phospholipids such as DSPC); (3) structural lipids (e.g., sterols such as cholesterol); and (4) PEGylated lipids.In one example by Semple et al. (Nature Biotech. 2010 28:172-176, the entire content of which is incorporated by reference), the lipid nanoparticle formulation is composed of the following molar ratio: 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA. As another example, altering the composition of cationic lipids can more efficiently deliver siRNA to various antigen-presenting cells (Basha et al., Mol Ther. 2011 19:2186-2200, incorporated by reference in its entirety).

[0117] In certain embodiments, lipid nanoparticles comprise cationic lipids and neutral lipids.In certain embodiments, LNPs comprise cationic lipids and DSPC-substituted moieties.In certain embodiments, LNPs comprise cationic lipids and fatty acids.In certain embodiments, LNPs comprise cationic lipids and oleic acid.In certain embodiments, LNPs comprise cationic lipids and oleic acid analogues.

[0118] In certain embodiments, lipid nanoparticle formulations comprise cationic lipids, neutral lipids and structured lipids.In certain embodiments, LNPs comprise cationic lipids, fatty acids and structured lipids.In certain embodiments, LNPs comprise cationic lipids, oleic acid and structured lipids.In certain embodiments, LNPs comprise cationic lipids, oleic acid analogs and structured lipids.In certain embodiments, LNPs comprise cationic lipids, fatty acids and sterols.In certain embodiments, LNPs comprise cationic lipids, oleic acid and sterols.In certain embodiments, LNPs comprise cationic lipids, oleic acid and cholesterol.

[0119] In certain embodiments, lipid nanoparticles comprise cationic lipid, neutral lipid and PEGylated lipid.In certain embodiments, LNP formulations comprise cationic lipid, neutral lipid and PEG-OH lipid.In certain embodiments, lipid nanoparticles comprise cationic lipid, fatty acid and PEG-OH lipid.In certain embodiments, lipid nanoparticles comprise cationic lipid, oleic acid and PEG-OH lipid.In certain embodiments, lipid nanoparticles comprise cationic lipid, oleic acid analog and PEG-OH lipid.

[0120] In certain embodiments, the lipid nanoparticles comprise a cationic lipid, a neutral lipid (e.g., a phospholipid or a fatty acid), a structured lipid, and a PEG-lipid. In certain embodiments, the lipid nanoparticle formulation comprises a cationic lipid, a neutral lipid (e.g., a phospholipid or a fatty acid), a structured lipid, and a PEG-OH lipid. In certain embodiments, the LNPs comprise a cationic lipid, a neutral lipid (e.g., a phospholipid or a fatty acid), and a structured lipid. In certain embodiments, the LNPs comprise a cationic lipid, a fatty acid (e.g., an oleic acid or an analog), a structured lipid, and a PEG-OH lipid. In certain embodiments, the LNPs comprise a cationic lipid, a fatty acid (e.g., an oleic acid or an analog), a structured lipid, and a PEG-OH lipid. In certain embodiments, the LNPs comprise a cationic lipid, an oleic acid, a structured lipid (e.g., a sterol), and a PEG-OH lipid. In certain embodiments, the LNPs comprise a cationic lipid, an oleic acid, and a structured lipid (e.g., cholesterol). In certain embodiments, the LNPs comprise one or more cationic or neutral lipids, a fatty acid (e.g., an oleic acid), and a PEG-lipid. In certain embodiments, the LNP comprises one or more cationic or neutral lipids, fatty acids (eg, oleic acid), and PEG-OH lipids.

[0121] In some embodiments, the LNP comprises a fatty acid. In certain embodiments, the fatty acid is a monounsaturated fatty acid. In certain embodiments, the fatty acid is a polyunsaturated fatty acid. In some embodiments, the LNP comprises oleic acid. In certain embodiments, the LNP comprises one or more cationic or neutral lipids and a fatty acid (e.g., oleic acid). In certain embodiments, the LNP comprises one or more cationic or neutral lipids and oleic acid. In certain embodiments, when the LNP comprises oleic acid, the LNP does not comprise a phospholipid. In certain embodiments, when the LNP comprises oleic acid, the LNP does not comprise DSPC. In certain embodiments, when the LNP comprises a fatty acid, the LNP does not comprise a phospholipid. In certain embodiments, when the LNP comprises a fatty acid, the LNP does not comprise DSPC.

[0122] In some embodiments, the LNP comprises, by molar percentage, 35-45% cationic lipid, 40-50% cationic lipid, 45-55% cationic lipid, 50-60% cationic lipid, and / or 55-65% cationic lipid. In some embodiments, the ratio of lipid to nucleic acid (e.g., mRNA) in the lipid nanoparticle can be 5:1-20:1, 10:1-25:1, 15:1-40:1, 20:1-30:1, 25:1-50:1, 30:1-60:1, and / or at least 40:1.

[0123] In some embodiments, the ratio of PEG in the LNP can be increased or decreased, and / or the carbon chain length of the alkyl portion of the PEG lipid can be varied from C8 to C18 (8 to 18 carbons) to alter the pharmacokinetics and / or biodistribution of the LNP. In certain embodiments, the LNP can contain 0.1% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.0% to 3.0%, 2.5% to 5.0%, and / or 3.0% to 6.0% PEGylated lipid, relative to other components. As a non-limiting example, the LNPs may contain 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.0% to 3.0%, 2.5% to 5.0%, and / or 3.0% to 6.0% PEG-c-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) relative to the cationic lipid, DSPC, and cholesterol. In some embodiments, PEG-c-DOMG can be replaced with a PEG lipid, such as, but not limited to, PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol), DMG-PEG (1,2-dimyristoyl-sn-glycerol) and / or PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid can be selected from any lipid known in the art, such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA. In certain embodiments, the lipid nanoparticle does not contain a PEG lipid. In certain embodiments, the lipid nanoparticle contains a PEG lipid, such as a PEG-OH lipid. The incorporation of a PEG-OH lipid into the nanoparticle formulation can improve the pharmacokinetics and / or biodistribution of LNP. For example, the incorporation of a PEG-OH lipid into the nanoparticle formulation can reduce the ABC effect.In certain embodiments, the LNPs comprise a lipid molar ratio of PEG-OH lipids to other components (e.g., cationic lipids, neutral lipids, and structural lipids) of 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.0% to 5.0%, 2.5% to 5.0%, and / or 3.0% to 6.0%, each possibility representing a separate embodiment of the present invention.

[0124] In some embodiments, the LNP comprises at least one lipid. In certain embodiments, the lipid is selected from cationic / ionizable lipids, neutral lipids (e.g., fatty acids and phospholipids), PEG lipids (e.g., PEG-OH lipids, methyl PEG (mPEG) lipids, ethyl PEG lipids, and other derivatized PEG-lipid conjugates), and structural lipids (e.g., sterols). The lipid can be selected from DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids, and aminoalcohol lipids. In some embodiments, the lipid can be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and aminoalcohol lipids. The amino alcohol cationic lipid may be the lipid described in U.S. Patent Publication No. US 2013 / 0150625, which is incorporated herein by reference in its entirety, and / or may be produced by the method described therein. Non-limiting examples of cationic lipids include 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 1 in U.S. Patent Publication No. US 2013 / 0150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (compound 2 in U.S. Patent Publication No. US 20130150625); 2-amino-3-[ (9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (compound 3 of US 2013 / 0150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 4 of US 2013 / 0150625); or any pharmaceutically acceptable salt or stereoisomer thereof.Each possibility represents a separate embodiment of the present invention.

[0125] The lipid nanoparticle formulations may contain lipids, in particular ionizable cationic lipids, such as 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (DLin-KC2-DMA). 3 19), and may further comprise neutral lipids (e.g., phospholipids or fatty acids), structural lipids (e.g., sterols such as cholesterol), and molecules that can reduce particle aggregation, such as PEG or PEGylated lipids (e.g., mPEG lipids or PEG-OH lipids). In certain embodiments, the formulation does not comprise a PEG lipid.

[0126] In some embodiments, the LNP formulation consists essentially of 20-60% cationic lipid; 5-25% neutral lipid; 25-55% sterol; and 0.1-15% PEG lipid. In some embodiments, the LNP formulation consists essentially of 20-60% cationic lipid; 5-25% neutral lipid; 25-55% sterol; and 0.1-15% mPEG lipid. In some embodiments, the LNP formulation consists essentially of a molar ratio of 20-60% cationic lipid; 5-25% neutral lipid; and 25-55% sterol. In certain embodiments, the neutral lipid is a fatty acid. In certain embodiments, the neutral lipid is oleic acid or an analog thereof. In certain embodiments, the PEG lipid is an mPEG lipid or a PEG-OH lipid.

[0127] In some embodiments, the LNPs comprise (i) 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (DLin-KC2-DMA), in a molar ratio of 20-60% cationic lipid; 5-25% neutral lipid; 25-55% sterol; and 0.1-15% PEG lipid. 3 19); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE, and SM; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA. Each possibility represents a separate embodiment of the present invention.

[0128] In some embodiments, the LNPs comprise (i) 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (DLin-KC2-DMA), in a molar ratio of 20-60% cationic lipid; 5-25% DSPC-substituted component; 25-55% structural lipid; and 0.1-15% PEG lipid. 3 19); (ii) a neutral lipid (e.g., various phospholipids or fatty acids) as a DSPC-substituting component; (iii) a structural lipid (e.g., a sterol such as cholesterol); and (iv) a PEG-lipid or PEG-OH lipid (e.g., PEG-DMG or PEG-cDMA). Each possibility represents a separate embodiment of the present invention.

[0129] In some embodiments, the LNP comprises 25% to 75% cationic lipid on a molar basis, such as 35% to 65%, 45% to 65%, 60%, 57.5%, 50%, or 40% of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (DLin-KC2-DMA). 3 19), each possibility representing a separate embodiment of the present invention.

[0130] In some embodiments, the LNPs comprise 0.5% to 15% neutral lipid on a molar basis, e.g., 3% to 12%, 5% to 10%, or 15%, 10%, or 7.5% on a molar basis. In certain embodiments, the neutral lipid is a phospholipid. In certain embodiments, the neutral lipid is a DSPC-substituted component (e.g., a phospholipid other than DSPC, 5%, or a fatty acid). In certain embodiments, the neutral lipid is a fatty acid (e.g., oleic acid or an analog thereof). Other examples of neutral lipids include, but are not limited to, POPC, DPPC, DOPE, and SM. In some embodiments, the LNPs comprise 0.5% to 15% fatty acid on a molar basis, e.g., 3% to 12%, 5% to 10%, or 15%, 10%, or 7.5% on a molar basis. In some embodiments, the LNPs comprise 0.5% to 15% oleic acid on a molar basis, e.g., 3% to 12%, 5% to 10%, or 15%, 10%, or 7.5% oleic acid on a molar basis. In some embodiments, the LNPs comprise 0.5% to 15% oleic acid on a molar basis, eg, 3-12%, 5-10%, or 15%, 10%, or 7.5% on a molar basis.

[0131] In some embodiments, the formulation contains 5% to 50% structured lipid on a molar basis, e.g., 15% to 45%, 20% to 40%, 41%, 38.5%, 35%, or 31% on a molar basis. In some embodiments, the formulation contains 5% to 50% sterol on a molar basis, e.g., 15% to 45%, 20% to 40%, 41%, 38.5%, 35%, or 31% on a molar basis. In some other embodiments, the formulation contains about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or about 45% on a molar basis. A non-limiting example of a sterol is cholesterol.

[0132] In some embodiments, the LNPs comprise 0.5% to 20% on a molar basis, e.g., 0.5 to 10%, 0.5 to 5%, 1.5%, 0.5%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, or 5% PEG or PEGylated lipids on a molar basis. In some embodiments, the PEG or PEGylated lipids comprise PEG molecules with an average molecular weight of 2,000 Da. In some embodiments, the PEG or PEGylated lipids comprise PEG molecules with an average molecular weight of less than 2,000, e.g., about 1,500 Da, about 1,000 Da, or about 500 Da. Non-limiting examples of PEGylated lipids include PEG-distearoylglycerol (PEG-DMG) (also referred to herein as Cmpd422), PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005), incorporated herein by reference in its entirety). As described herein, any PEG or PEGylated lipid can be a PEG-OH lipid. In some embodiments, the LNP contains 0.5% to 20% on a molar basis, e.g., 0.5-10%, 0.5-5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% PEG-OH lipid on a molar basis.

[0133] In some embodiments, the LNPs comprise, on a molar basis, 25-75% cationic lipid, 0.5-15% neutral lipid; 5-50% structural lipid, and 0.5-20% PEG or PEGylated lipid. In some embodiments, the LNPs comprise, on a molar basis, 25-75% cationic lipid, 0.5-15% neutral lipid; 5-50% structural lipid, and 0.5-20% PEG-OH lipid. In some embodiments, the LNPs comprise, on a molar basis, 25-75% cationic lipid, 0.5-15% neutral lipid, and 5-50% structural lipid. In some embodiments, the LNPs contain 25-75% of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (DLin-KC2-DMA). 3 19).

[0134] In some embodiments, the LNPs comprise, on a molar basis, 35-65% cationic lipids, 3-12% neutral lipids, 15-45% structural lipids, and 0.5-10% PEG or PEGylated lipids. In some embodiments, the LNPs comprise, on a molar basis, 35-65% cationic lipids, 3-12% neutral lipids, 15-45% structural lipids, and 0.5-10% PEG-OH lipids. In some embodiments, the LNPs comprise, on a molar basis, 35-65% cationic lipids, 3-12% neutral lipids, and 15-45% structural lipids. In some embodiments, the LNPs contain 35-65% of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (DLin-KC2-DMA). 3 19), each possibility representing a separate embodiment of the present invention.

[0135] In some embodiments, the LNPs comprise, on a molar basis, 45-65% cationic lipids, 5-10% neutral lipids, 25-40% structural lipids, and 0.5-10% PEG or PEGylated lipids. In some embodiments, the LNPs comprise, on a molar basis, 45-65% cationic lipids, 5-10% neutral lipids, 25-40% structural lipids, and 0.5-10% PEG-OH lipids. In some embodiments, the LNPs comprise, on a molar basis, 45-65% cationic lipids, 5-10% neutral lipids, and 25-40% structural lipids. In some embodiments, the LNPs are composed of 45-65% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (DLin-KC2-DMA). 3 19), each possibility representing a separate embodiment of the present invention.

[0136] In some embodiments, the LNPs comprise, on a molar basis, 60% cationic lipids, 7.5% neutral lipids, 31% structural lipids, and 1.5% PEG or PEGylated lipids. In some embodiments, the LNPs comprise, on a molar basis, 60% cationic lipids, 7.5% neutral lipids, 31% structural lipids, and 1.5% PEG-OH lipids. In some embodiments, the LNPs comprise, on a molar basis, 60% cationic lipids, 9% neutral lipids, and 31% structural lipids. In some embodiments, the LNPs comprise, on a molar basis, 60% cationic lipids, 9% neutral lipids, and 31% structural lipids. In some embodiments, the LNPs comprise 60% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (DLin-KC2-DMA). 3 19), each possibility representing a separate embodiment of the present invention.

[0137] In some embodiments, the LNPs comprise, on a molar basis, 50% cationic lipids, 10% neutral lipids, 38.5% structural lipids, and 1.5% PEG or PEGylated lipids. In some embodiments, the LNPs comprise, on a molar basis, 50% cationic lipids, 10% neutral lipids, 38.5% structural lipids, and 1.5% PEG-OH lipids. In some embodiments, the LNPs comprise, on a molar basis, 50% cationic lipids, 10% neutral lipids, and 40% structural lipids. In some embodiments, the LNPs comprise, on a molar basis, 50% cationic lipids, 10% neutral lipids, and 40% structural lipids. In some embodiments, the LNPs comprise 50% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (DLin-KC2-DMA). 3 19), each possibility representing a separate embodiment of the present invention.

[0138] In some embodiments, the LNPs comprise, on a molar basis, 40% cationic lipids, 15% neutral lipids, 40% structural lipids, and 5% PEG or PEGylated lipids. In some embodiments, the LNPs comprise, on a molar basis, 40% cationic lipids, 15% neutral lipids, 40% structural lipids, and 5% PEG-OH lipids. In some embodiments, the LNPs comprise, on a molar basis, 40% cationic lipids, 20% neutral lipids, and 40% structural lipids. In some embodiments, the LNPs comprise, on a molar basis, 40% cationic lipids, 20% neutral lipids, and 40% structural lipids. In some embodiments, the LNPs comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (DLin-KC2-DMA). 3 19), each possibility representing a separate embodiment of the present invention.

[0139] In some embodiments, the LNPs, on a molar basis, comprise 57.2% cationic lipids, 7.1% neutral lipids, 34.3% sterol, and 1.4% PEG or PEGylated lipids. In some embodiments, the LNPs, on a molar basis, comprise 57.2% cationic lipids, 7.1% neutral lipids, 34.3% structural lipids, and 1.4% PEG-OH lipids. In some embodiments, the LNPs, on a molar basis, comprise 57.2% cationic lipids, 8.5% neutral lipids, and 34.3% structural lipids. In some embodiments, the LNP is 57.2% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (DLin-KC2-DMA). 3 19), each possibility representing a separate embodiment of the present invention.

[0140] In some embodiments, the LNPs consist essentially of a lipid mixture with a molar ratio of 20-70% cationic lipid, 5-45% neutral lipid, 20-55% structural lipid, and 0.1-15% PEGylated lipid. In some embodiments, the LNPs consist essentially of a lipid mixture with a molar ratio of 20-70% cationic lipid, 5-45% neutral lipid (e.g., phospholipid or fatty acid), 20-55% structural lipid, and 0.1-15% PEG-OH lipid. In some embodiments, the LNPs consist essentially of a lipid mixture with a molar ratio of 20-70% cationic lipid, 5-45% neutral lipid (e.g., phospholipid or fatty acid), 20-55% structural lipid (e.g., sterol), and 0.1-15% PEG-OH lipid. In some embodiments, the LNPs consist essentially of a lipid mixture in a molar ratio of 20-70% cationic lipid; 5-45% neutral lipid (e.g., phospholipid or fatty acid); and 20-55% structural lipid (e.g., sterol). In some embodiments, the LNPs consist essentially of a lipid mixture in a molar ratio of 20-70% cationic lipid; 5-45% fatty acid (e.g., oleic acid or analog thereof); 20-55% structural lipid (e.g., sterol); and 0.1-15% PEG-OH lipid. In some embodiments, the LNPs consist essentially of a lipid mixture in a molar ratio of 20-70% cationic lipid; 5-45% fatty acid (e.g., oleic acid or analog thereof); and 20-55% structural lipid (e.g., sterol). In some embodiments, the LNPs consist essentially of a lipid mixture in a molar ratio of 20-70% cationic lipid, 5-45% oleic acid, 20-55% structural lipid (e.g., sterol), and 0.1-15% PEG-OH lipid. In some embodiments, the LNPs consist essentially of a lipid mixture in a molar ratio of 20-70% cationic lipid, 5-45% oleic acid, and 20-55% structural lipid (e.g., sterol).

[0141] Non-limiting examples of lipid nanoparticle compositions and methods for their preparation are described, for example, in Jayarama et al. (2012), Angew. Chem. Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety).

[0142] In some embodiments, the LNPs may comprise a cationic lipid, a PEG lipid (e.g., a PEG-OH lipid), and optionally a neutral lipid (e.g., a phospholipid or a fatty acid). In some embodiments, the LNPs may comprise a cationic lipid, a PEG lipid (e.g., a PEG-OH lipid), and a structural lipid (e.g., a sterol), and optionally a neutral lipid (e.g., a phospholipid or a fatty acid).

[0143] The lipid nanoparticles described herein may comprise two or more components (e.g., lipids) excluding the payload. In certain embodiments, the LNP comprises two or more components (e.g., lipids) excluding the payload. In certain embodiments, the lipid nanoparticle comprises five components (e.g., lipids) excluding the payload. In certain embodiments, the LNP comprises five components (e.g., lipids) excluding the payload.

[0144] In some embodiments, the LNPs described herein comprise four-component lipid nanoparticles. The four-component LNPs may comprise four different lipids selected from any of those described herein. The four components do not comprise a payload. The lipid nanoparticles may comprise cationic lipids, neutral lipids, PEG lipids, and structured lipids. In certain embodiments, the lipid nanoparticles comprise cationic lipids, fatty acids, PEG lipids, and structured lipids. In certain embodiments, the lipid nanoparticles comprise cationic lipids, fatty acids, PEG-OH lipids, and structured lipids. Each possibility represents a separate embodiment of the present invention.

[0145] In some embodiments, the LNPs described herein can be three-component lipid nanoparticles. Three-component LNPs can comprise three different lipids as described herein. Lipid nanoparticles can comprise cationic lipids, neutral lipids (e.g., phospholipids or fatty acids) and structured lipids. In certain embodiments, lipid nanoparticles comprise cationic lipids, fatty acids and structured lipids. In certain embodiments, lipid nanoparticles comprise cationic lipids, phospholipids and structured lipids.

[0146] In one embodiment, the LNP formulation can be formulated according to the methods described in International Publication Nos. WO 2011127255 or WO 2008103276, the contents of each of which are incorporated herein by reference in their entireties. Non-limiting examples include the LNP formulations described in WO 2011127255 and / or WO 2008103276, the contents of each of which are incorporated herein by reference in their entireties.

[0147] In one embodiment, the lipid nanoparticles can be formulated according to the methods described in U.S. Patent Application No. 2013 / 0156845 or International Publication No. WO 2013 / 093648 or WO 2012024526, each of which is incorporated herein by reference in its entirety.

[0148] The lipid nanoparticles described herein can be prepared in a sterile environment by the systems and / or methods described in U.S. Patent Publication No. 20130164400, which is incorporated herein by reference in its entirety.

[0149] In one embodiment, the LNP formulations can be formulated in nanoparticles, such as the nucleic acid-lipid nanoparticles described in US Pat. No. 8,492,359, the contents of which are incorporated herein by reference in their entirety.

[0150] As non-limiting examples, lipid nanoparticles can include one or more active or therapeutic agents (e.g., RNA); one or more cationic lipids comprising about 50 mol% to about 85 mol% of all lipids present in the particle; one or more neutral lipids comprising about 13 mol% to about 49.5 mol% of all lipids present in the particle; and one or more structural lipids that inhibit particle aggregation, comprising about 0.5 mol% to about 2 mol% of all lipids present in the particle.

[0151] In one embodiment, the LNP formulation can be formulated according to the methods described in International Publication No. WO 2011127255 or WO 2008103276, the contents of each of which are incorporated herein by reference in their entirety. Non-limiting examples include the LNP formulations described in WO 2011127255 and / or WO 2008103276, the contents of each of which are incorporated herein by reference in their entirety. In one embodiment, the LNP formulations described herein can include a polycationic composition. Non-limiting examples include polycationic compositions selected from Formulas 1-60 of U.S. Patent Publication No. 20050222064, the contents of which are incorporated herein by reference in their entirety.

[0152] In some embodiments, the LNP comprises the lipid KL52 (an amino lipid described in U.S. Patent Publication No. 2012 / 0295832, the entire contents of which are expressly incorporated herein by reference). The activity and / or safety of LNP administration (measured by testing one or more of ALT / AST, white blood cell count, and cytokine induction) can be improved by incorporating such lipids. LNPs comprising KL52 can be administered intravenously and / or in one or more doses. In some embodiments, administration of LNPs comprising KL52 results in comparable or improved mRNA and / or protein expression compared to LNPs comprising MC3.

[0153] As a non-limiting example, the LNP may comprise a cationic peptide or polypeptide, such as, but not limited to, polylysine, polyornithine, and / or polyarginine, and a cationic peptide, as described in International Publication No. WO 2012013326 or U.S. Patent Publication No. 20130142818, each of which is incorporated herein by reference in its entirety. In some embodiments, the lipid nanoparticle comprises a neutral lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0154] The nanoparticle composition can be relatively uniform. The polydispersity index can be used to indicate the uniformity of the nanoparticle composition, for example, the particle size distribution of the nanoparticle composition. A low polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be from about 0.10 to about 0.20, or from about 0.05 to about 0.15, or less than about 0.1, or less than about 0.15, each possibility representing a separate embodiment of the present invention.

[0155] The zeta potential of a nanoparticle composition can be used to indicate the interfacial conductivity potential of the composition. For example, the zeta potential indicates the surface charge of the nanoparticle composition. Nanoparticle compositions with a relatively low positive or negative charge at physiological pH are generally desirable because more highly charged species can interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV. Each possibility represents a separate embodiment of the present invention.

[0156] The encapsulation efficiency of a therapeutic agent indicates the amount of therapeutic agent encapsulated or bound to a nanoparticle composition after preparation, compared to the initial amount provided. A high encapsulation efficiency is desirable (e.g., near 100%). The encapsulation efficiency can be measured by measuring the amount of therapeutic agent in a solution containing the nanoparticle composition before or after disrupting the nanoparticle composition with one or more organic solvents or surfactants. Fluorescence can be used to measure the amount of free therapeutic agent (e.g., nucleic acid) in solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%. In certain embodiments, the encapsulation efficiency can be at least 95%. Each possibility represents a separate embodiment of the present invention.

[0157] Nanoparticle compositions may optionally include one or more coatings.For example, nanoparticle compositions can be formulated into capsules, films, or tablets with coatings.The capsules, films, or tablets containing the compositions described herein can have any useful size, tension, hardness, or density.

[0158] In some embodiments, such LNPs are synthesized using methods that involve a microfluidic mixer. Exemplary microfluidic mixers may include, but are not limited to, Slit Interdigital Microstructured Mixers, including micromixers manufactured by Microinnova (Allerheiligen bei Wilden, Austria), and / or staggered herringbone micromixers (SHM). (Zhigaltsev, IV et al., Bottom-up design and synthesis of limit-size lipid nanoparticle systems with aqueous and triglyceride centers using millisecond microfluidic mixing have been published (Langmuir. 2012. 28:3633-40; Belliveau, NM et al., Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA. Molecular Therapy-Nucleic Acids. 2012. 1:e37; Chen, D. et al., Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc. 2012). 134(16):6948-51).

[0159] In some embodiments, the method for producing LNPs using SHM further comprises mixing at least two input streams, wherein the mixing occurs via microstructure-induced chaotic advection (MICA). This method allows fluid flow through channels in a herringbone pattern, resulting in rotational flow and folding the fluids around each other. This method can also include a surface for fluid mixing, where the surface changes direction while the fluid circulates. Methods for purifying LNPs using SHM include those described in U.S. Application Publication Nos. 2004 / 0262223 and 2012 / 0276209, each of which is incorporated herein by reference in its entirety.

[0160] In one embodiment, lipid nanoparticles can be formulated using, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or a Caterpillar (CPMM) or an Impinging jet (IJMM) from Institut fur Mikrotechnik Mainz GmbH (Mainz, Germany).

[0161] In one embodiment, lipid nanoparticles are produced using microfluidic technology (see Whitesides, George M. The Origins and the Future of Microfluidics. Nature, 2006 442: 368-373; and Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651, which are incorporated herein by reference in their entirety).By way of non-limiting example, controlled microfluidic formulation comprises the passive method for mixing the flow by constant pressure in microchannels at low Reynolds number (see, for example, Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651, which are incorporated herein by reference in their entirety).

[0162] In one embodiment, therapeutic nucleic acids (e.g., mRNA) can be formulated in lipid nanoparticles created using a micromixer chip, such as, but not limited to, a micromixer chip from Harvard Apparatus (Holliston, Massachusetts) or Dolomite Microfluidics (Royston, UK). Micromixer chips can be used for rapid mixing of two or more fluid streams with a splitting and recombination mechanism.

[0163] cationic lipids The cationic lipids useful in the present invention are neutral in circulation, but become positively charged after endosomal acidification. The positive charge on LNPs can promote binding to negatively charged cell membranes and improve cellular uptake. Cationic lipids can also bind to negatively charged lipids, inducing a non-duplex structure that promotes intracellular delivery. Cationic lipids suitable for use in the manufacture of LNPs described herein can be ionizable cationic lipids described herein. Cationic lipids can be prepared according to the methods shown in the examples, or according to methods known or derivable by those skilled in the art.

[0164] In some embodiments, the LNP may comprise, by molar percentage, 35-45% cationic lipid, 40-50% cationic lipid, 45-55% cationic lipid, 50-60% cationic lipid, and / or 55-65% cationic lipid. In some embodiments, the ratio of lipid to nucleic acid (e.g., mRNA) in the lipid nanoparticle may be 5:1-20:1, 10:1-25:1, 15:1-40:1, 20:1-30:1, 25:1-50:1, 30:1-60:1, and / or at least 40:1.

[0165] Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N---(N',N'dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1-(2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE).

[0166] Additionally, many commercial products of cationic lipids are available that can be used in any of the described embodiments. These include, for example, LIPOFECTIN® (a commercially available cationic liposome containing DOTMA and 1,2-dioleoyl-sn-3 phosphoethanolamine (DOPE) from GIBCO / BRL, Grand Island, NY); LIPOFECT Amine® (a commercially available cationic liposome containing N-(1-(2,3 dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE) from GIBCO / BRL); and Transfect AM® (a commercially available cationic lipid containing dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol from Promega Corp., Madison, WI). The following lipids are cationic and have a positive charge below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0167] In certain embodiments, the cationic lipid for use in any of the described embodiments is independently amino lipid.Suitable amino lipids include those described in WO2010 / 054401 and WO2012 / 016184, which are incorporated herein by reference in their entirety. Representative amino lipids include, but are not limited to, 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane The cationic lipids include chloride salts (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA). In some of the described embodiments, the cationic lipid has the following structure: [ka] [During the ceremony, R1 and R2 are the same or different and independently represent optionally substituted C 10 -C 24 Alkyl, optionally substituted C 10 -C 24 Alkenyl, optionally substituted C 10 -C 24 Alkynyl or optionally substituted C 10 -C24 It is acyl; R3 and R4 are the same or different and independently represent optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl, or R3 and R4 can combine to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen; R5 is absent or present, and when present, is hydrogen or C1-C6 alkyl; m, n, and p are the same or different and independently 0 or 1, with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; Y and Z are the same or different and independently O, S, or NH. In one embodiment, R1 and R2 are each linoleyl, and the amino lipid is a dilinoleyl amino lipid. In one embodiment, the amino lipid is a dilinoleyl amino lipid. In various other embodiments, the cationic lipid has the following structure: [ka] [During the ceremony, R1 and R2 are independently selected from the group consisting of H and C1-C3 alkyl; R3 and R4 are independently selected from the group consisting of alkyl groups having from about 10 to about 20 carbon atoms, wherein at least one of R3 and R4 contains at least two unsaturated moieties (e.g., R3 and R4 are, for example, dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl). or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof. In a preferred embodiment, R3 and R4 are both linoleyl. R3 and R4 can contain at least three unsaturated moieties (e.g., R3 and R4 can be, for example, dodecatrienyl, tetradecatrienyl, hexadecatrienyl, linolenyl, and icosatrienyl).

[0168] In some embodiments, the cationic lipid has the following structure: [ka] [During the ceremony, R1 and R2 are independently selected to be H or C1-C3 alkyl; R3 and R4 are independently selected to be alkyl groups having from about 10 to about 20 carbon atoms, where at least one of R4 and R4 contains at least two unsaturated moieties. or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof. In one embodiment, R3 and R4 are both the same, for example, in some embodiments, R3 and R4 are both linoleyl (i.e., C18), etc. In another embodiment, R3 and R4 are different, for example, in some embodiments, R3 is tetradecatrienyl (C14) and R4 is linoleyl (C18). In a preferred embodiment, the cationic lipid of the present invention is symmetrical, i.e., R3 and R4 are the same. In another preferred embodiment, both R3 and R4 comprise at least two unsaturated moieties. In some embodiments, R3 and R4 are independently selected from dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl and icosadienyl. In a preferred embodiment, R3 and R4 are both linoleyl. In some embodiments, R4 and R4 comprise at least three unsaturated moieties and are independently selected from, for example, dodecatrienyl, tetradecatrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.

[0169] In various embodiments, the cationic lipid has the formula: [ka] [During the ceremony, X aa is the formula -NR N -CR 1 R 2a D-amino acid or L-amino acid residue having -C(C=O)-, or a peptide or a compound of the formula -{NR N ---CR 1 R 2 ---(C=O)} n a peptide of amino acid residues having the formula: R 1 is independently, for each occurrence, a non-hydrogen, substituted or unsubstituted side chain of an amino acid; R 2 and R N is independently, at each occurrence, an organic group containing hydrogen, carbon, oxygen, nitrogen, sulfur, and hydrogen atoms or any combination thereof and having 1 to 20 carbon atoms; C (1-5) Alkyl, cycloalkyl, cycloalkylalkyl, C (3-5) Alkenyl, C (3-5) Alkynyl, C (1-5) Alkanoyl, C (1-5) Alkanoyloxy, C (1-5) Alkoxy, C (1-5) Alkoxy-C (1-5) Alkyl, C (1-5) Alkoxy-C (1-5) Alkoxy, C (1-5) Alkyl-amino-C (1-5) Alkyl-, C (1-5) Dialkyl-amino-C (1-5) Alkyl-, nitro-C (1-5) Alkyl, Cyano-C (1-5) Alkyl, aryl-C (1-5) Alkyl, 4-biphenyl-C (1-5) is alkyl, carboxyl or hydroxyl; Z is NH, O, S, -CH2S-, -CH2S(O)- or an organic linker consisting of 1 to 40 atoms selected from hydrogen, carbon, oxygen, nitrogen and sulfur atoms (preferably, Z is NH or O); R x and R yare independently (i) a lipophilic terminus derived from a lipid (which may be naturally occurring or synthetic), a phospholipid, a glycolipid, a triacylglycerol, a glycerophospholipid, a sphingolipid, a ceramide, a sphingomyelin, a cerebroside, or a ganglioside, the terminus of which may optionally comprise a steroid; (ii) an amino acid terminal group selected from hydrogen, hydroxyl, amino, and an organic protecting group; or (iii) a substituted or unsubstituted C (3-22) Alkyl, C (6-12) Cycloalkyl, C (6-12) Cycloalkyl-C (3-22) Alkyl, C (3-22) Alkenyl, C (3-22) Alkynyl, C (3-22) Alkoxy or C (6-12) -Alkoxy-C (3-22) Contains alkyl; R x and R y one of which is a lipophilic end as defined above and the other is an amino acid end group, or R x and R y are both lipophilic ends; R x and R y At least one of the groups has one or more biodegradable groups (e.g., -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, -C(R 5 )=N-, -N=C(R 5 )--, -C(R 5 )=NO-, -ON=C(R 5 )-, -C(O)(NR 5 )-, -N(R 5 )C(O)-, -C(S)(NR 5 )-, -N(R 5 )C(O)-, -N(R 5 )C(O)N(R 5 )-, -OC(O)O-, --OSi(R 5 )2O-, -C(O)(CR 3 R 4 )C(O)O-, -OC(O)(CR 3 R 4 )C(O)- or [ka] (In the formula, R 11 is C2-C8 alkyl or alkenyl and R in each instance 5 is independently H or alkyl; 3 and R 4 are independently H, halogen, OH, alkyl, alkoxy, --NH, alkylamino, or dialkylamino; or R 3 and R 4 together with the carbon atom to which they are directly attached form a cycloalkyl group (in a preferred embodiment, R 3 and R 4 are independently H or C1-C4 alkyl; R x and R y each independently optionally has one or more carbon-carbon double bonds) may be interrupted by or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0170] In some embodiments, the cationic lipid is one of the following, or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof: [ka] [During the ceremony, R1 and R2 are independently alkyl, alkenyl, or alkynyl, each of which may be optionally substituted; R3 and R4 are independently C1-C6 alkyl, or R3 and R4 can together form an optionally substituted heterocyclic ring.

[0171] Representative useful dilinoleyl amino lipids have the formula: [ka] wherein n is 0, 1, 2, 3, or 4. It has the following characteristics.

[0172] In one embodiment, the cationic lipid is DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMA (the DLin-K-DMA described above, where n is 2).

[0173] In one embodiment, the cationic lipid has the following structure: [ka] [During the ceremony, R1 and R2 are each independently, in each occurrence, optionally substituted C 10 -C 30 Alkyl, optionally substituted C 10 -C 30 Alkenyl, optionally substituted C 10 -C 30 Alkynyl or optionally substituted C 10 -C 30 is an acyl or linker-ligand; R3 is H, optionally substituted C1-C 10 Alkyl, optionally substituted C-C 10 Alkenyl, optionally substituted C-C 10 R3 is an alkynyl, alkyl heterocycle, alkyl phosphate, alkyl phosphorothioate, alkyl phosphorodithioate, alkyl phosphonate, alkylamine, hydroxyalkyl, ω-aminoalkyl, ω-(substituted) aminoalkyl, ω-phosphoalkyl, ω-thiophosphoalkyl, optionally substituted polyethylene glycol (PEG, molecular weight 100-40K), optionally substituted mPEG (molecular weight 120-40K), heteroaryl, or heterocycle, or linker-ligand, e.g., in some embodiments, R3 is (CH3)2N(CH2) n where n is 1, 2, 3 or 4; E is O, S, N(Q), C(O), OC(O), C(O)O, N(Q)C(O), C(O)N(Q), (Q)N(CO)O, O(CO)N(Q), S(O), NS(O)2N(Q), S(O)2, N(Q)S(O)2, SS, O=N, aryl, heteroaryl, cyclic ring or heterocycle, e.g., -C(O)O, where - is the point of attachment to R3; Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkyl, or ω-thiophosphoalkyl. or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0174] In certain embodiments, the cationic lipid has the following structure: [ka] [During the ceremony, E is O, S, N(Q), C(O), N(Q)C(O), C(O)N(Q), (Q)N(CO)O, O(CO)N(Q), S(O), NS(O)2N(Q), S(O)2, N(Q)S(O)2, SS, O=N, aryl, heteroaryl, cyclic ring or heterocycle; Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkyl, or ω-thiophosphoalkyl; R1 and R2 and R x are each independently, at each occurrence, H, optionally substituted C-C 10 Alkyl, optionally substituted C 10 -C 30 Alkyl, optionally substituted C 10 -C 30 Alkenyl, optionally substituted C 10 -C 30 Alkynyl, optionally substituted C 10 -C 30 acyl or linker-ligand, where R, R and R x At least one of them is not H; R3 is H, optionally substituted C1-C 10 Alkyl, optionally substituted C-C 10 Alkenyl, optionally substituted C-C 10 alkynyl, alkyl heterocycle, alkyl phosphate, alkyl phosphorothioate, alkyl phosphorodithioate, alkyl phosphonate, alkylamine, hydroxyalkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkyl, ω-thiophosphoalkyl, optionally substituted polyethylene glycol (PEG, molecular weight 100-40K), optionally substituted mPEG (molecular weight 120-40K), heteroaryl, or heterocycle, or linker-ligand; n is 0, 1, 2, or 3. or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0175] In another embodiment, the cationic lipid has one of the following structures: [ka] [ka]

[0176] In some embodiments, the cationic lipid is DLin-M-C3-DMA, MC3, or M-C3, and is described in WO 2010 / 054401 and WO 2010 / 144740 A1.

[0177] In different embodiments, the cationic lipid has one of the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0178] In another embodiment, the cationic lipid has the following structure: [ka] [During the ceremony, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen, optionally substituted C-C 30 Alkyl, optionally substituted C7-C 30 Alkenyl and optionally substituted C-C 30 alkynyl selected from the group consisting of: However, (a)R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R8 at least two of these are not hydrogen, and (b) R is not hydrogen. 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 at least two of which are in the 1,3, 1,4 or 1,5 positions relative to one another; X is selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl; R 9 , R 10 and R 11 are independently selected from the group consisting of hydrogen, optionally substituted C-C alkyl, optionally substituted C-C alkenyl, and optionally substituted C-C alkynyl, with the proviso that R 9 , R 10 and R 11 One of the may not be present; n and m are each independently 0 or 1. or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0179] In certain embodiments, the cationic lipid has the following structure: [ka]

[0180] In one embodiment, the cationic lipid has the following structure: [ka] [During the ceremony, R1 is independently selected from -(CH2)2-N(R)2, -(CH2)2-N(R)-(CH2)2-N(R)2, where R is independently -H, C 6-40 Alkyl, C 6-40 Alkenyl and C 6-40alkynyl with the proviso that -N(R)2 is not NH2; R2 is C 6-40 Alkyl, C 6-40 Alkenyl or C 6-40 is alkynyl; m is 0 or 1. or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0181] In a more specific embodiment, the cationic lipid is: [ka] The compound has a structure selected from:

[0182] In another embodiment, the cationic lipid has the following structure: [ka] [During the ceremony, R' is absent, hydrogen or alkyl; R 1 and R 2 Regarding (i)R 1 and R 2 are each independently an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle; (ii)R 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted heterocyclic ring; or (iii)R 1 and R 2 one of which is an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle, and the other is (a) the adjacent nitrogen atom and (b) (R) adjacent to the nitrogen atom. a a 4- to 10-membered heterocyclic ring or heteroaryl substituted with a group; R in each instance is independently -(CR3 R 4 )-and; R in each case 3 and R 4 is independently H, OH, alkyl, alkoxy, -NH2, alkylamino, or dialkylamino; or R 3 and R 4 together with the carbon atom to which they are directly attached form a cycloalkyl group, where carbon C * In each chain attached to, at most three R are cycloalkyl; A dashed line to Q is absent or a bond; When there is no dashed line to Q, Q is either absent or -O-, -NH-, -S-, -C(O)O-, -OC(O)-, -C(O)N(R 4 )-, -N(R 5 )C(O)-, -SS-, -OC(O)O-, -ON=C(R 5 )-, -C(R 5 )=NO-, -OC(O)N(R 5 )-, -N(R 5 )C(O)N(R 5 )-, -N(R 5 )C(O)O-, -C(O)S-, -C(S)O- or -C(R 5 )=NOC(O)-; or When the dashed line to Q is a bond, (i) b is 0, and (ii) Q and it (C * ) and the adjacent tertiary carbons form a substituted or unsubstituted monocyclic or bicyclic heterocyclic group having 5 to 10 ring atoms; Q 1 and Q 2 are each independently absent, -O-, -S-, -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, -C(O)(NR 5 )-, -N(R 5 )C(O)-, -C(S)(NR 5 )-, -N(R 5 )C(O)-, -N(R 5 )C(O)N(R 5)- or -OC(O)O-; Q 3 and Q 4 are each independently H, -(CR 3 R 4 )-, aryl or cholesterol moiety; A in each case 1 , A 2 , A 3 and A 4 are independently -(CR 5 R 5 -CR 5 =CR 5 )-and; R in each case 5 is independently H or alkyl; M 1 and M 2 are each independently a biodegradable group; The biodegradable group is -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, -C(R 5 )=N-, -N=C(R 5 )-, -C(R 5 )=NO-, -ON=C(R 5 )-, -C(O)(NR 5 )-, -N(R 5 )C(O)-, -C(S)(NR 5 )-, -N(R 5 )C(O)-, -N(R 5 )C(O)N(R 5 )-, -OC(O)O-, -OSi(R 5 )2O-, -C(O)(CR 3 R 4 )C(O)O- and -OC(O)(CR 3 R 4 )C(O)-; Z is absent, alkylene, or -OP(O)(OH)-O-; Each --- attached to Z is Q when Z is not present. 3 and Q 4 is any bond such that the bond is not a direct covalent bond; a is 1, 2, 3, 4, 5 or 6; b is 0, 1, 2 or 3; c, d, e, f, i, j, m, n, q and r are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; g and h are each independently 0, 1, or 2; k and l are each independently 0 or 1, where at least one of k and l is 1; o and p are each independently 0, 1, or 2; where (i) a compound comprising the following moiety: [ka] (In the formula, ---- is any bond; Q 3 and Q 4 are each independently formed into a star ( * ) is separated from the tertiary carbon atom marked (not including) or its salts.

[0183] In more specific embodiments, the cationic lipid is selected from the following compounds or salts thereof (eg, pharmaceutically acceptable salts thereof): [ka] [ka] [During the ceremony, m, n, o, and p are each independently 1 to 25, with the proviso that: (i) In structures (II), (IV), (VI), and (VII), both m and p are 4 or greater; (ii) In structures (VIII), (X), (XII), (XIV), (XVI), (XVIII), (XXI), and (XXIII), m is 4 or greater; (iii) In structures (VIII), (IX), (XII) and (XIII), p is 8 or greater (e.g., 12 or 14 or greater).

[0184] In yet another more specific embodiment, the cationic lipid has the structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0185] In some embodiments, the cationic lipid has the following structure: [ka] [During the ceremony, R1 is C 5-30 Alkyl, C 5-20 Alkenyl, -R * selected from the group consisting of -YR'', -YR'', and -R''M'R'; R2 and R3 are independently H, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is C 3-6 Carbocycle, -(CH2)nQ, -(CH2) n CHQR, -CHQR, -CQ(R)2 and unsubstituted C 1-6and wherein Q is selected from the group consisting of carbocycle, heterocycle, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC selected from -(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR and -C(R)N(R)2C(O)OR, wherein each n is independently selected from 1, 2, 3, 4 and 5; Each R5 is independently 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl and H; Each R6 is independently 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl and H; M and M' are independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl and H; R8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; Each R is independently 1-3 Alkyl, C 2-3 selected from the group consisting of alkenyl and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, -R * selected from the group consisting of YR″, -YR″ and H; Each R'' is independently C 3-14 Alkyl and C 3-14 alkenyl; Each R * independently, C 1-12 Alkyl and C 2-12 alkenyl; Each Y is independently 3-6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13. or a salt or isomer thereof.

[0186] In yet another embodiment, the cationic lipid is selected from the following compounds: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0187] In some embodiments, the cationic lipid has the following structure: [ka] [During the ceremony, R' is absent, hydrogen or C1-C4 alkyl; R 1 and R 2 Regarding (i)R 1 and R 2 are each independently an optionally substituted alkyl, alkenyl, alkynyl, cycloalkylalkyl, heterocycle, or R 10 and; (ii)R 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted heterocyclic ring; or (iii)R 1 and R 2 one of which is an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle, and the other is (a) the adjacent nitrogen atom and (b) (R) adjacent to the nitrogen atom. a forming a 4- to 10-membered heterocyclic ring or heteroaryl with the group; R in each instance is independently -(CR 3 R 4 )-and; R in each case 3 and R 4 are independently H, halogen, OH, alkyl, alkoxy, -NH2, R 10, alkylamino or dialkylamino; R in each case 10 are independently selected from PEG and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide], and poly(amino acid), wherein (i) the PEG or polymer is linear or branched, (ii) the PEG or polymer is polymerized with n subunits, (iii) n is a number average degree of polymerization from 10 to 200 units, and (iv) the compound of said formula contains at most two R 10 having a group; A dashed line to Q is absent or a bond; When there is no dashed line to Q, Q is absent or is -O-, -NH-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R 4 )-, -N(R 5 )C(O)-, -SS-, -OC(O)O-, -ON=C(R 5 )-, -C(R 5 )=NO-, -OC(O)N(R 5 )-, -N(R 5 )C(O)N(R 5 )-, -N(R 5 )C(O)O-, -C(O)S-, -C(S)O- or -C(R 5 )=NOC(O)-; or When the dashed line to Q is a bond, (i) b is 0, and (ii) Q and its adjacent tertiary carbon (C * ) form a substituted or unsubstituted monocyclic or bicyclic heterocycle having 5 to 10 ring atoms; R in each case 5 are independently H or C1-C4 alkyl; M 1 and M 2 are each independently -OC(O)-, -C(O)O-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-, -SS-, -C(R 5 )=N-, -N=C(R 5)-, -C(R 5 )=NO-, -ON=C(R 5 )-, -C(O)(NR 5 )-, -N(R 5 )C(O)-, -C(S)(NR 5 )-, -N(R 5 )C(O)-, -N(R 5 )C(O)N(R 5 )-, -OC(O)O-, -OSi(R 5 )2O-, -C(O)(CR 3 R 4 )C(O)O- and -OC(O)(CR 3 R 4 a biodegradable group selected from: C(O)—; [ka] (In the formula, R 11 is a C2-C8 alkyl or alkenyl) and; R in each case z are independently C1-C8 alkyl; a is 1, 2, 3, 4, 5 or 6; b is 0, 1, 2 or 3; L 1 and L 2 are each independently C1-C5 alkylene or C2-C5 alkenylene; X and Y are each independently alkylene or alkenylene; Z 1 and Z 2 are each independently C8-C 14 Alkyl or C8-C 14 alkenyl, wherein the alkenyl group optionally comprises a double bond and Z 1 or Z 2 may be substituted with one or two fluorine atoms at the α-position of the double bond between the ends of Z 1 and Z 2 At least one end of M is bounded by at least 8 carbon atoms 1 or M 2 separated from the group or a salt thereof.

[0188] In yet another embodiment, the cationic lipid is selected from the following compounds: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

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[0189] In one embodiment, the cationic lipid has the structure of one of the following compounds and salts thereof: [ka] [ka] [ka] [ka]

[0190] In a further embodiment, the cationic lipid has the structure of one of the following compounds and salts thereof: [ka] [ka] [ka] [ka] [ka] [ka]

[0191] In certain embodiments, the cationic lipid has the structure of one of the following compounds and salts thereof: [ka]

[0192] Further representative cationic lipids include, but are not limited to:

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[0193] In another embodiment, the cationic lipid has the following structure: [ka] [During the ceremony, R' is absent, hydrogen or C1-C4 alkyl; R 1 and R 2 Regarding R' is absent, hydrogen or alkyl; R 1 and R 2 Regarding (i)R 1 and R 2 are each independently an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, heterocycle, or R 10 and; (ii)R 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted heterocyclic ring; or (iii)R 1 and R 2 is an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle, and the other is (a) an adjacent nitrogen atom and (b) an (R) adjacent to the nitrogen atom. a a 4- to 10-membered heterocyclic ring or heteroaryl having a group; R in each instance is independently -(CR 3 R 4 )-and; R in each case 3 and R4 are independently hydrogen, OH, alkyl, alkoxy, -NH2, R 10 , alkylamino or dialkylamino; R in each case 10 are independently selected from PEGs and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide], and poly(amino acid), wherein (i) the PEG or polymer is linear or branched, (ii) the PEG or polymer is polymerized with n subunits, (iii) n is a number average degree of polymerization from 10 to 200 units, and (iv) the compound of the formula contains at most two R 10 having a group; A dashed line to Q is absent or a bond; When there is no dashed line to Q, Q is absent or is -O-, -NH-, -S-, -C(O)-, -C(O)O, -OC(O)-, -C(O)N(R 4 )-, -N(R 5 )C(O)-, -SS-, -OC(O)O-, -ON=C(R 5 )-, -C(R 5 )=NO-, -OC(O)N(R 5 )-, -N(R 5 )C(O)N(R 5 )-, -N(R 5 )C(O)O-, -C(O)S-, -C(S)O- or -C(R 5 )=NOC(O)-; or When the dashed line to Q is a bond, (i) b is 0, and (ii) Q and its adjacent tertiary carbon (C * ) form a substituted or unsubstituted monocyclic or bicyclic heterocyclic group having 5 to 10 ring atoms; R in each case 5 are independently hydrogen or alkyl; X and Y each independently represent -(CR 6 R 7 ) c - and; R in each case 6and R 7 are independently hydrogen, OH, alkyl, alkoxy, -NH2, alkylamino, or dialkylamino; M 1 and M 2 are each independently a biodegradable group; a is 1, 2, 3, 4, 5 or 6; b is 0, 1, 2 or 3; c in each instance is independently 2 to 10; Z 1 and Z 2 are each independently: (i) C3-C 10 cycloalkyl, (ii) C-C 10 cycloalkyl(C1-C6 alkyl) or (iii) [ka] (In the formula, R 8 and R 9 each of which is a C2-C8 alkyl is] or a salt or isomer thereof.

[0194] In yet another embodiment, the cationic lipid is selected from the following compounds: [ka] [ka] [ka] [ka] [ka]

[0195] In one embodiment, the cationic lipid has Formula I: [ka] [During the ceremony, L 1 or L 2 One of the is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O-, and L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, ,NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or a direct bond; R a is H or C1-C 12 is alkyl; R 1a and R 1b is, in each occurrence independently: (a) H or C-C 12 alkyl, or (b) R 1a is H or C1-C 12 alkyl, and R 1b together with the carbon atom to which it is attached, the adjacent R 1b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 2a and R 2b is, in each occurrence independently: (a) H or C-C 12 alkyl, or (b) R 2a is H or C1-C 12 alkyl, and R 2btogether with the carbon atom to which it is attached, the adjacent R 2b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 3a and R 3b is, in each occurrence independently: (a) H or C-C 12 alkyl, or (b) R 3a is H or C1-C 12 alkyl, and R 3b together with the carbon atom to which it is attached, the adjacent R 3b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 4a and R 4b is, in each occurrence independently: (a) H or C-C 12 alkyl, or (b) R 4a is H or C1-C 12 alkyl, and R 4b together with the carbon atom to which it is attached, the adjacent R 4b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 5 and R 6 are each independently methyl or cycloalkyl; R 7 is, in each occurrence, independently H or C-C 12 is alkyl; R 8 and R 9 are each independently an unsubstituted C-C 12 alkyl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocyclic ring containing one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; e is 1 or 2; x is 0, 1, or 2. or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0196] In some embodiments of Formula (I), L 1 and L 2 are independently —O(C═O)— or —(C═O)O—.

[0197] In certain embodiments of Formula (I), R 1a , R 2a , R 3a or R 4a At least one of the following is C1-C 12 alkyl or L 1 or L 2 At least one of R is -O(C=O)- or -(C=O)O-. 1a and R 1b is not isopropyl when a is 6 or n-butyl when a is 8.

[0198] In further embodiments of Formula (I), R 1a , R 2a , R 3a or R 4a At least one of the following is C1-C 12 alkyl or L 1 or L 2 at least one of is -O(C=O)- or -(C=O)O-; R 1a and R 1b is not isopropyl when a is 6 or n-butyl when a is 8.

[0199] In other embodiments of Formula (I), R 8 and R 9 are each independently an unsubstituted C-C 12 alkyl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocyclic ring containing one nitrogen atom.

[0200] In certain embodiments of Formula (I), L 1 or L 2 Either of L can be -O(C=O)- or a carbon-carbon double bond. 1 and L 2 can each be —O(C═O)— or can each be a carbon-carbon double bond.

[0201] In some embodiments of Formula (I), L 1 or L 2 One of L is -O(C=O)-. 1 and L 2 Both are -O(C=O)-.

[0202] In some embodiments of Formula (I), L 1 or L 2 One of the groups is -(C=O)O-. 1 and L 2 Both are —(C═O)O—.

[0203] In some other embodiments of Formula (I), L 1 or L 2 One of L is a carbon-carbon double bond. 1 and L 2 Both are carbon-carbon double bonds.

[0204] In still other embodiments of Formula (I), L 1 or L 2 One of them is -O(C=O)- and L 1 or L 2 and the other is —(C═O)O—. In a further embodiment, L 1 or L 2 One of them is -O(C=O)- and L 1 or L 2 The other of L is a carbon-carbon double bond. 1 or L 2 One of them is -(C=O)O- and L 1or L 2 The other is a carbon-carbon double bond.

[0205] As used throughout this specification, a "carbon-carbon" double bond refers to a bond having the following structure: [ka] [In the formula, R a and R b is, in each occurrence, independently H or a substituent. For example, in some embodiments, R a and R b is, in each occurrence independently, H, C-C 12 Alkyl or cycloalkyl, e.g., H or C1-C 12 It is alkyl.

[0206] In other embodiments, the lipid compound of formula (I) has the following formula (Ia): [ka] It has the following structure.

[0207] In other embodiments, the lipid compound of formula (I) has the following formula (Ib): [ka] It has the following structure.

[0208] In yet another embodiment, the lipid compound of formula (I) has the following formula (Ic): [ka] It has the following structure.

[0209] In certain embodiments of lipid compounds of Formula (I), a, b, c, and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c, and d are each independently an integer from 8 to 12 or from 5 to 9. In some particular embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In further embodiments, a is 3. In still other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In further embodiments, a is 7. In still other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In further embodiments, a is 11. In still other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In further embodiments, a is 15. In still other embodiments, a is 16.

[0210] In some other embodiments of Formula (I), b is 1. In other embodiments, b is 2. In further embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In further embodiments, b is 7. In still other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In further embodiments, b is 11. In still other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In further embodiments, b is 15. In still other embodiments, b is 16.

[0211] In some further embodiments of Formula (I), c is 1. In other embodiments, c is 2. In further embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In further embodiments, c is 7. In still other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In further embodiments, c is 11. In still other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In further embodiments, c is 15. In still other embodiments, c is 16.

[0212] In certain other embodiments of Formula (I), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In further embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In further embodiments, d is 7. In still other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In further embodiments, d is 11. In still other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In further embodiments, d is 15. In still other embodiments, d is 16.

[0213] In some other various embodiments of Formula (I), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same, and b and c are the same.

[0214] The sum of a and b and the sum of c and d in Formula (I) are factors that can be varied to obtain a lipid of Formula (I) with desired properties. In one embodiment, a and b are selected so that their sum is an integer in the range of 14 to 24. In another embodiment, c and d are selected so that their sum is an integer in the range of 14 to 24. In further embodiments, the sum of a and b and the sum of c and d are the same. For example, in some embodiments, the sum of a and b and the sum of c and d are both the same integer, which may be in the range of 14 to 24. In further embodiments, a, b, c, and d are selected so that the sum of a and b and the sum of c and d is 12 or greater.

[0215] In some embodiments of Formula (I), e is 1. In other embodiments, e is 2.

[0216] R in formula (I) 1a , R 2a , R 3a and R 4a The substituents of R are not particularly limited. 1a , R 2a , R 3a and R 4a is, at each occurrence, H. In certain other embodiments, R 1a , R 2a , R 3a and R 4a At least one of the following is C1-C 12 In certain other embodiments, R 1a , R 2a , R 3a and R 4a At least one of R is C-C alkyl. 1a , R 2a , R 3a and R 4a At least one of is C1-C6 alkyl. In some of the above embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0217] In certain embodiments of Formula (I), R 1a , R 1b , R 4a and R 4b In each case, C1-C 12 It is alkyl.

[0218] In further embodiments of Formula (I), R 1b , R 2b , R 3b and R 4b At least one of is H or R 1b , R 2b , R 3b and R 4b is H in each case.

[0219] In certain embodiments of Formula (I), R 1b together with the carbon atom to which it is attached, the adjacent R 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond. 4b together with the carbon atom to which it is attached, the adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0220] R in formula (I) 5 and R 6 The substituents of R are not particularly limited. 5 or R 6 One or both of R 5 or R 6 One or both of is cycloalkyl, e.g., cyclohexyl. In these embodiments, the cycloalkyl can be substituted or unsubstituted. In certain other embodiments, the cycloalkyl is C-C 12 It is substituted with alkyl, for example, tert-butyl.

[0221] In the above embodiment of Formula I, R7 In certain embodiments, at least one R 7 is H. In some other embodiments, R 7 is H in each occurrence. In certain other embodiments, R 7 is C1-C 12 It is alkyl.

[0222] In certain other embodiments of the above embodiments of Formula (I), R 8 or R 9 One of R is methyl. 8 and R 9 Both of the are methyl.

[0223] In some different embodiments of Formula (I), R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocyclic ring. 8 and R 9 together with the nitrogen atom to which they are attached form a 5-membered heterocyclic ring, for example, a pyrrolidinyl ring.

[0224] In various different embodiments, the lipid of formula (I) has one of the structures shown in Table 1 below. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7]

[0225] In some embodiments, the cationic lipid has Formula II: [ka] [During the ceremony, L 1 or L 2 One of the is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O-, and L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, ,NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or a direct bond; G 1 is C1-C2 alkylene, -(C=O)-, -O(C=O)-, -SC(=O)-, -NR a C(=O)- or a direct bond; G 2 -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NR a -or is a direct bond; G 3 is C1-C6 alkylene; R a is H or C1-C 12 is alkyl; R 1aand R 1b is, in each occurrence independently: (a) H or C-C 12 alkyl; or (b) R 1a is H or C1-C 12 alkyl, and R 1b together with the carbon atom to which it is attached, the adjacent R 1b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 2a and R 2b is, in each occurrence independently: (a) H or C-C 12 alkyl; or (b) R 2a is H or C1-C 12 alkyl, and R 2b together with the carbon atom to which it is attached, the adjacent R 2b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 3a and R 3b is, in each occurrence, independently: (a): H or C-C 12 alkyl; or (b) R 3a is H or C1-C 12 alkyl, and R 3b together with the carbon atom to which it is attached, the adjacent R 3b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 4a and R 4b is, in each occurrence independently: (a) H or C-C 12 alkyl; or (b) R 4a is H or C1-C 12 alkyl, and R 4b together with the carbon atom to which it is attached, the adjacent R 4b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 5 and R 6 are each independently H or methyl; R 7 is C4-C 20is alkyl; R 8 and R 9 are each independently C1-C 12 alkyl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocyclic ring; a, b, c, and d are each independently an integer from 1 to 24; x is 0, 1, or 2. or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0226] In some embodiments of Formula (II), L 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, or a direct bond. 1 and G 2 Each is independently —(C═O)— or a direct bond. 1 and L 2 are each independently —O(C═O)—, —(C═O)O—, or a direct bond; G 1 and G 2 is each independently —(C═O)— or a direct bond.

[0227] In some different embodiments of Formula (II), L 1 and L 2 are each independently -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a -, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a , -OC(=O)NR a -, -NR a C(=O)O-, -NR a S(O) x NR a -, -NR aS(O) x -or-S(O) x NR a -It is.

[0228] In another embodiment of the above embodiment of Formula (II), the lipid compound has the following Formula (IIA) or Formula (IIB): [ka]

[0229] In some embodiments of Formula (II), the lipid compound has the formula (IIA): In other embodiments, the lipid compound has the formula (IIB):

[0230] In any of the above embodiments of Formula (II), L 1 or L 2 One of the groups is —O(C═O)—. For example, in some embodiments, L 1 and L 2 Each of is —O(C═O)—.

[0231] In some different embodiments of Formula (II), L 1 or L 2 One of the groups is —(C═O)O—. For example, in some embodiments, L 1 and L 2 Each of is —(C═O)O—.

[0232] Different embodiments of formula (II), L 1 or L 2 As used herein, a "direct bond" refers to a bond that is bonded to a group (e.g., L 1 or L 2 ) is absent. For example, in some embodiments, L 1 and L 2 Each of is a direct bond.

[0233] In other different embodiments of formula (II), R 1a and R 1bFor at least one of the 1a is H or C1-C 12 alkyl, and R 1b together with the carbon atom to which it is attached, the adjacent R 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0234] In yet another different embodiment of formula (II), R 4a and R 4b For at least one of the 4 is H or C1-C 12 alkyl, and R 4b together with the carbon atom to which it is attached, the adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0235] In a further embodiment of Formula (II), R 2a and R 2b For at least one of the 2a is H or C1-C 12 alkyl, and R 2b together with the carbon atom to which it is attached, the adjacent R 2b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0236] In other different embodiments of formula (II), R 3a and R 3b For at least one of the 3a is H or C1-C 12 alkyl, and R 3b together with the carbon atom to which it is attached, the adjacent R 3b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0237] In various other embodiments of Formula (II), the lipid compound has the following Formula (IIC) or Formula (IID): [ka] wherein e, f, g, and h each independently represent an integer of 1 to 12. It has one of the following.

[0238] In some embodiments of Formula (II), the lipid compound has the formula (IIC): In other embodiments, the lipid compound has the formula (IID):

[0239] In various embodiments of Formula (IIC) or Formula (IID), e, f, g, and h are each independently an integer from 4 to 10.

[0240] In certain embodiments of Formula (II), a, b, c, and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c, and d are each independently an integer from 8 to 12 or 5 to 9. In some particular embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In further embodiments, a is 3. In still other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In further embodiments, a is 7. In still other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In further embodiments, a is 11. In still other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In further embodiments, a is 15. In still other embodiments, a is 16.

[0241] In some embodiments of Formula (II), b is 1. In other embodiments, b is 2. In further embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In further embodiments, b is 7. In still other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In further embodiments, b is 11. In still other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In further embodiments, b is 15. In still other embodiments, b is 16.

[0242] In some embodiments of Formula (II), c is 1. In other embodiments, c is 2. In further embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In further embodiments, c is 7. In still other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In further embodiments, c is 11. In still other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In further embodiments, c is 15. In still other embodiments, c is 16.

[0243] In some particular embodiments of Formula (II), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In further embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In further embodiments, d is 7. In still other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In further embodiments, d is 11. In still other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In further embodiments, d is 15. In still other embodiments, d is 16.

[0244] In some embodiments of Formula (II), e is 1. In other embodiments, e is 2. In further embodiments, e is 3. In yet other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In further embodiments, e is 7. In still other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In further embodiments, e is 11. In still other embodiments, e is 12.

[0245] In some embodiments of Formula (II), f is 1. In other embodiments, f is 2. In further embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In further embodiments, f is 7. In still other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In further embodiments, f is 11. In still other embodiments, f is 12.

[0246] In some embodiments of Formula (II), g is 1. In other embodiments, g is 2. In further embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In further embodiments, g is 7. In still other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In further embodiments, g is 11. In still other embodiments, g is 12.

[0247] In some embodiments of Formula (II), h is 1. In other embodiments, e is 2. In further embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In further embodiments, h is 7. In still other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In further embodiments, h is 11. In still other embodiments, h is 12.

[0248] In various other embodiments of Formula (II), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same, and b and c are the same.

[0249] The sum of a and b and c and d in Formula (II) are factors that can be varied to obtain lipids with desired properties. In one embodiment, a and b are selected so that their sum is an integer ranging from 14 to 24. In another embodiment, c and d are selected so that their sum is an integer ranging from 14 to 24. In further embodiments, the sum of a and b and the sum of c and d are the same. For example, in some embodiments, the sum of a and b and the sum of c and d are both the same integer, which can range from 14 to 24. In further embodiments, a, b, c, and d are selected so that the sum of a and b and the sum of c and d are 12 or greater.

[0250] R in formula (II) 1a , R 2a , R 3a and R 4a The substituents of R are not particularly limited. 1a , R 2a , R 3a and R 4a At least one of R is H. In certain embodiments, 1a , R 2a , R 3a and R 4a is, at each occurrence, H. In certain other embodiments, R 1a , R 2a , R 3a and R 4a At least one of the following is C1-C 12 In certain other embodiments, R 1a , R 2a , R 3a and R 4a At least one of R is C-C alkyl. 1a , R 2a , R 3a and R 4a At least one of is C1-C6 alkyl. In some of the above embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0251] In certain embodiments of Formula (II), R 1a , R 1b , R 4a and R 4b In each case, C1-C 12 It is alkyl.

[0252] In a further embodiment of Formula (II), R 1b , R 2b , R 3b and R 4b At least one of is H or R 1b , R 2b , R 3b and R 4b is H in each case.

[0253] In certain embodiments of Formula (II), R 1b together with the carbon atom to which it is attached, the adjacent R 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond. 4b together with the carbon atom to which it is attached, the adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0254] In the above embodiment, R of formula (II) 5 and R 6 The substituents of R are not particularly limited. 5 or R 6 is methyl. In other embodiments, R 5 or R 6 Each of is methyl.

[0255] R in formula (II) 7 The substituents of R are not particularly limited. 7 is C6-C 16 In some other embodiments, R 7 is C6-C9 alkyl. In some of these embodiments, R7 -(C=O)OR b , -O(C=O)R b , -C(=O)R b , -OR b , -S(O) x R b , -S-SR b , -C(=O)SR b , -SC(=O)R b , -NR a R b , -NR a C(=O)R b , -C(=O)NR a R b , -NR a C(=O)NR a R b , -OC(=O)NR a R b , -NR a C(=O)OR b , -NR a S(O) x NR a R b , -NR a S(O) x R b or -S(O) x NR a R b wherein R a is H or C1-C 12 alkyl; b is C1-C 15 alkyl; and x is 0, 1, or 2. For example, in some embodiments, R 7 -(C=O)OR b or -O(C=O)R b is replaced by

[0256] In some of the above embodiments of formula (II), R b is branch C1-C 16 For example, in some embodiments, R b has one of the following structures: [ka]

[0257] In certain other embodiments of the above embodiment of Formula (II), R 8 or R 9 One of R is methyl. 8 and R 9 Both of the are methyl.

[0258] In some different embodiments of Formula (II), R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocyclic ring. 8 and R 9 together with the nitrogen atom to which they are attached form a 5-membered heterocyclic ring, e.g., a pyrrolidinyl ring. In some different embodiments of the above, R 8 and R 9 together with the nitrogen atom to which they are attached form a six-membered heterocyclic ring, for example, a piperazinyl ring.

[0259] In yet another embodiment of the lipid of formula (II), G 3 is a C2-C4 alkylene, for example, a C3 alkylene. In various different embodiments, the lipid compound has one of the structures shown in Table 2 below. [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15]

[0260] In some other embodiments, the cationic lipid has formula (III): [ka] [During the ceremony, L 1 or L 2 One of the is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O-, and L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, ,NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or a direct bond; G 1 and G 2 are each independently an unsubstituted C-C 12 Alkylene or C1-C 12 alkenylene; G 3 is C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1-C12 is alkyl; R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 is alkenyl; R 3 is H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and; R 4 is C1-C 12 is alkyl; R 5 is H or C1-C6 alkyl; x is 0, 1, or 2. or a pharmaceutically acceptable salt or stereoisomer thereof.

[0261] In some of the above embodiments of formula (III), the lipid has the following formula (IIIA) or formula (IIIB): [ka] [During the ceremony, A is a 3- to 8-membered cycloalkyl or cycloalkylene ring; R 6 is, in each occurrence, independently H, OH, or C-C 24 is alkyl; n is an integer ranging from 1 to 15. It has one of the following.

[0262] In some of the above embodiments of Formula (III), the lipid has Formula (IIIA), and in other embodiments, the lipid has Formula (IIIB).

[0263] In other embodiments of Formula (III), the lipid has the following Formula (IIIC) or Formula (IIID): [ka] wherein y and z are each independently an integer ranging from 1 to 12. It has one of the following.

[0264] In any of the above embodiments of formula (III), L 1 or L 2 One of the groups is —O(C═O)—. For example, in some embodiments, L 1 and L 2 Each of L is -O(C=O)-. 1 and L 2 is each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of is —(C═O)O—.

[0265] In some different embodiments of Formula (III), the lipid has the following formula (IIIE) or formula (IIIF): [ka] It has one of the following.

[0266] In some of the above embodiments of Formula (III), the lipid has the following Formula (IIIG), Formula (IIIH), Formula (IIII), or Formula (IIIJ): [ka] It has one of the following.

[0267] In some of the above embodiments of Formula (III), n is an integer ranging from 2 to 12, e.g., from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0268] In some other embodiments of the above embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0269] In some of the above embodiments of formula (III), R 6 is H. In another embodiment of the above embodiment, R 6 is C1-C 24 In another embodiment, R 6 is OH.

[0270] In some embodiments of Formula (III), G 3 is unsubstituted. 3 is substituted. In various different embodiments, G 3 is a straight chain C1-C 24 Alkylene or straight chain C1-C 24 It is alkenylene.

[0271] In some other such embodiments of formula (III), R 1 or R 2 , or both of these, C6-C 24 For example, in some embodiments, R 1 and R 2 each independently have the structure: [ka] [During the ceremony, R 7a and R 7b is, in each occurrence, independently H or C-C 12 is alkyl; a is an integer ranging from 2 to 12; R 7a , R 7b and a are R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or from 8 to 12.

[0272] In some of the above embodiments of formula (III), at least one instance of R 7a is H. For example, in some embodiments, R 7a is H in each occurrence. In other different embodiments of the above, at least one occurrence of R 7b is C1-C8 alkyl. For example, in some embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0273] In different embodiments of formula (III), R 1 or R 2 , or both of these, with the following structure: [ka] It has.

[0274] In some of the above embodiments of formula (III), R 3 OH, CN, -C(=O)OR 4 , -OC(=O)R 4 or -NHC(=O)R 4 In some embodiments, R 4 is methyl or ethyl.

[0275] In various different embodiments, the cationic lipid has one of the structures shown in Table 3 below. [Table 16] [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] [Table 22] [Table 23]

[0276] In one embodiment, the cationic lipid has formula (IV): [ka] [During the ceremony, G 1 or G 2 one of which is, in each case, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -SS-, -C(=O)S-, SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, -N(R a )C(=O)N(R a )-, -OC(=O)N(R a )- or -N(R a )C(=O)O-, and G 1 or G 2 The other is, in each case, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -SS-, -C(=O)S-, -SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, -N(R a )C(=O)N(R a )-, -OC(=O)N(R a )- or -N(R a )C(=O)O- or a direct bond; L, in each occurrence, is ~O(C=O)-, where ~ represents a covalent bond to X; X is CR a and; When n is 1, Z is an alkyl, cycloalkyl, or monovalent moiety containing at least one polar functional group; or when n is greater than 1, Z is an alkylene, cycloalkylene, or polyvalent moiety containing at least one polar functional group; R a is, in each occurrence, independently: H, C-C 12 Alkyl, C1-C 12 Hydroxyalkyl, C1-C 12 Aminoalkyl, C1-C 12 Alkylaminylalkyl, C1-C 12 Alkoxyalkyl, C1-C 12 Alkoxycarbonyl, C1-C 12 Alkylcarbonyloxy, C1-C 12 Alkylcarbonyloxyalkyl or C1-C 12 is alkylcarbonyl; R, in each occurrence, is independently: (a) H or C-C 12 alkyl; or (b) R, together with the carbon atom to which it is attached, forms a carbon-carbon double bond with an adjacent R and the carbon atom to which it is attached; R 1 and R 2 In each case, the following structures are [ka] having; a 1 and a 2 is, in each occurrence, independently an integer from 3 to 12; b 1 and b 2 is, at each occurrence, independently 0 or 1; c 1 and c 2 is, in each occurrence, independently an integer from 5 to 10; d1 and d 2 is, in each occurrence, independently an integer from 5 to 10; y, in each occurrence, is independently an integer from 0 to 2; n is an integer ranging from 1 to 6; wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl, and alkylcarbonyl may be optionally substituted with one or more substituents. or a pharmaceutically acceptable salt or stereoisomer thereof.

[0277] In some embodiments of Formula (IV), G 1 and G 2 is each independently —O(C═O)— or —(C═O)O—.

[0278] In other embodiments of Formula (IV), X is CH.

[0279] In different embodiments of formula (IV), a 1 +b 1 +c 1 the sum of or a 2 +b 2 +c 2 The sum of these is an integer in the range of 12 to 26.

[0280] In still other embodiments of formula (IV), a 1 and a 2 are independently an integer from 3 to 10. For example, in some embodiments, a 1 and a 2 are independently an integer from 4 to 9.

[0281] In various embodiments of formula (IV), b 1 and b 2 is 0. In a different embodiment, b 1 and b 2 is 1.

[0282] In a further embodiment of formula (IV), c 1 , c 2 , d 1 and d 2 are independently an integer from 6 to 8.

[0283] In other embodiments of formula (IV), c 1 and c 2 is, in each occurrence, independently an integer from 6 to 10; and d 1 and d 2 is, in each occurrence, independently an integer from 6 to 10.

[0284] In other embodiments of formula (IV), c 1 and c 2 is, independently in each occurrence, an integer from 5 to 9; and d 1 and d 2 is, in each occurrence, independently an integer from 5 to 9.

[0285] In further embodiments of Formula (IV), when n is 1, Z is alkyl, cycloalkyl, or a monovalent moiety containing at least one polar functional group. In other embodiments, Z is alkyl.

[0286] In various embodiments of Formula (IV) above, R, at each occurrence, is independently: (a) H or methyl; or (b) R, together with the carbon atom to which it is attached, forms a carbon-carbon double bond with an adjacent R and the carbon atom to which it is attached. In certain embodiments, each R is H. In other embodiments, at least one R, together with the carbon atom to which it is attached, forms a carbon-carbon double bond with an adjacent R and the carbon atom to which it is attached.

[0287] In other embodiments of compounds of Formula (IV), R 1 and R 2 independently has one of the following structures: [ka]

[0288] In certain embodiments of Formula (IV), the compound has one of the following structures: [ka] [ka] [ka] [ka]

[0289] In yet another embodiment, the cationic lipid has the formula (V): [ka] [During the ceremony, G 1 or G 2 one of which is, in each case, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -SS-, -C(=O)S-, SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, -N(R a )C(=O)N(R a )-, -OC(=O)N(R a )- or -N(R a )C(=O)O-, and G 1 or G 2 The other is, in each case, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) y -, -SS-, -C(=O)S-, -SC(=O)-, -N(R a )C(=O)-, -C(=O)N(R a )-, -N(R a )C(=O)N(R a )-, -OC(=O)N(Ra )- or -N(R a )C(=O)O- or a direct bond; L, in each occurrence, is ~O(C=O)-, where ~ represents a covalent bond to X; X is CR a and; When n is 1, Z is an alkyl, cycloalkyl, or monovalent moiety containing at least one polar functional group; or when n is greater than 1, Z is an alkylene, cycloalkylene, or polyvalent moiety containing at least one polar functional group; R a is, in each occurrence, independently: H, C-C 12 Alkyl, C1-C 12 Hydroxyalkyl, C1-C 12 Aminoalkyl, C1-C 12 Alkylaminylalkyl, C1-C 12 Alkoxyalkyl, C1-C 12 Alkoxycarbonyl, C1-C 12 Alkylcarbonyloxy, C1-C 12 Alkylcarbonyloxyalkyl or C1-C 12 is alkylcarbonyl; R, at each occurrence, is independently: (a) H or C-C 12 alkyl; or (b) R, together with the carbon atom to which it is attached, forms a carbon-carbon double bond with an adjacent R and the carbon atom to which it is attached; R 1 and R 2 are, in each case, the following structures: [ka] having; R', at each occurrence, is independently H or C-C 12 is alkyl; a 1 and a 2 is, in each occurrence, independently an integer from 3 to 12; b 1 and b2 is, at each occurrence, independently 0 or 1; c 1 and c 2 is, in each occurrence, independently an integer from 2 to 12; d 1 and d 2 is, in each occurrence, independently an integer from 2 to 12; y, in each occurrence, is independently an integer from 0 to 2; n is an integer ranging from 1 to 6; where a 1 , a 2 , c 1 , c 2 , d 1 and d 2 is a 1 +c 1 +d 1 The sum of is an integer in the range of 18 to 30, and a 2 +c 2 +d 2 is selected so that the sum is an integer in the range of 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl, and alkylcarbonyl may optionally be substituted with one or more substituents. or a pharmaceutically acceptable salt or stereoisomer thereof.

[0290] In certain embodiments of Formula (V), G 1 and G 2 is each independently —O(C═O)— or —(C═O)O—.

[0291] In other embodiments of Formula (V), X is CH.

[0292] In some embodiments of Formula (V), a 1 +c 1 +d 1 The sum of is an integer in the range of 20 to 30, and a 2 +c2 +d 2 The sum of a is an integer ranging from 18 to 30. 1 +c 1 +d 1 The sum of is an integer in the range of 20 to 30, and a 2 +c 2 +d 2 The sum of a is an integer in the range of 20 to 30. In a further embodiment of formula (V), a 1 +b 1 +c 1 the sum of or a 2 +b 2 +c 2 The sum of a is an integer ranging from 12 to 26. 1 , a 2 , c 1 , c 2 , d 1 and d 2 is a 1 +c 1 +d 1 The sum of is an integer in the range of 18 to 28, and a 2 +c 2 +d 2 The sum is selected to be an integer in the range of 18 to 28.

[0293] In still other embodiments of Formula (V), a 1 and a 2 are independently an integer of 3 to 10, for example, an integer of 4 to 9.

[0294] In yet another embodiment of Formula (V), b 1 and b 2 is 0. In a different embodiment, b 1 and b 2 is 1.

[0295] In certain other embodiments of Formula (V), c 1 , c 2 , d 1 and d 2 are independently an integer from 6 to 8.

[0296] In other embodiments of Formula (V), when n is 1, Z is an alkyl or monovalent moiety containing at least one polar functional group; or when n is greater than 1, Z is an alkylene or polyvalent moiety containing at least one polar functional group.

[0297] In further embodiments of Formula (V), when n is 1, Z is alkyl, cycloalkyl, or a monovalent moiety containing at least one polar functional group. In other embodiments, Z is alkyl.

[0298] In other different embodiments of Formula (V), R, at each occurrence, is independently: (a) H or methyl; or (b) R, together with the carbon atom to which it is attached, forms a carbon-carbon double bond with an adjacent R and the carbon atom to which it is attached. For example, in some embodiments, each R is H. In other embodiments, at least one R, together with the carbon atom to which it is attached, forms a carbon-carbon double bond with an adjacent R and the carbon atom to which it is attached.

[0299] In further embodiments, each R' is H.

[0300] In certain embodiments of Formula (V), a 1 +c 1 +d 1 The sum of is an integer between 20 and 25, and 2 +c 2 +d 2 The sum of these is an integer in the range of 20 to 25.

[0301] In other embodiments of Formula (V), R 1 and R 2 independently has one of the following structures: [ka]

[0302] In further embodiments of Formula (V), the compound has one of the following structures: [ka] [ka] [ka]

[0303] In any of the above embodiments of Formula (IV) or Formula (V), n is 1. In other embodiments of the above embodiments of Formula (IV) or Formula (V), n is greater than 1.

[0304] In further embodiments of any of the above embodiments of Formula (IV) or Formula (V), Z is a monovalent or polyvalent moiety comprising at least one polar functional group. In some embodiments, Z is a monovalent moiety comprising at least one polar functional group. In other embodiments, Z is a polyvalent moiety comprising at least one polar functional group.

[0305] In further embodiments of either of the above embodiments of Formula (IV) or Formula (V), the polar functional group is a hydroxyl, alkoxy, ester, cyano, amido, amino, alkylaminyl, heterocyclyl, or heteroaryl functional group.

[0306] In any of the above embodiments of Formula (IV) or Formula (V), Z is hydroxyl, hydroxylalkyl, alkoxyalkyl, amino, aminoalkyl, alkylaminyl, alkylaminylalkyl, heterocyclyl, or heterocyclylalkyl.

[0307] In some other embodiments of Formula (IV) or Formula (V), Z has the following structure: [ka] [During the ceremony, R 5 and R 6 are independently H or C1-C6 alkyl; R7 and R 8 are independently H or C1-C6 alkyl, or R 7 and R 8 together with the nitrogen atom to which they are attached, form a 3- to 7-membered heterocyclic ring; x is an integer ranging from 0 to 6. It has.

[0308] In yet another embodiment of Formula (IV) or Formula (V), Z has the following structure: [ka] [During the ceremony, R 5 and R 6 are independently H or C1-C6 alkyl; R 7 and R 8 are independently H or C1-C6 alkyl, or R 7 and R 8 together with the nitrogen atom to which they are attached, form a 3- to 7-membered heterocyclic ring; x is an integer ranging from 0 to 6. It has.

[0309] In yet another embodiment of formula (IV) or formula (V), Z has the structure [ka] [During the ceremony, R 5 and R 6 are independently H or C1-C6 alkyl; R 7 and R 8 are independently H or C1-C6 alkyl, or R 7 and R 8 together with the nitrogen atom to which they are attached, form a 3- to 7-membered heterocyclic ring; x is an integer ranging from 0 to 6. It has.

[0310] In some other embodiments of Formula (IV) or Formula (V), Z is hydroxylalkyl, cyanoalkyl, or alkyl substituted with one or more ester or amide groups.

[0311] For example, in any of the above embodiments of Formula (IV) or Formula (V), Z has one of the following structures: [ka]

[0312] In other embodiments of Formula (IV) or Formula (V), ZL has one of the following structures: [ka] [ka]

[0313] In other embodiments, ZL has one of the following structures: [ka]

[0314] In still other embodiments, X is CH and ZL has one of the following structures: [ka]

[0315] In various different embodiments, the cationic lipid has one of the structures shown in Table 4 below. [Table 24]

[0316] In one embodiment, the cationic lipid has the following formula (VI): [ka] [During the ceremony, L 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a -, -NR a C(=O)O- or a direct bond; G 1 is C1-C2 alkylene, -(C=O)-, -O(C=O)-, -SC(=O)-, -NR a C(=O)- or a direct bond; G 2 -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NR a -or is a direct bond; G 3 is C1-C6 alkylene; R a is H or C1-C 12 is alkyl; R 1a and R 1b is, in each occurrence, independently: (a) H or C-C 12 alkyl; or (b) R 1a is H or C1-C 12 alkyl, and R 1b together with the carbon atom to which it is attached, the adjacent R 1b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 2a and R 2b is, in each occurrence, independently: (a) H or C-C 12 alkyl; or (b) R 2a is H or C1-C 12 alkyl, and R 2b together with the carbon atom to which it is attached, the adjacent R 2band together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 3a and R 3b is, in each occurrence, independently: (a): H or C-C 12 alkyl; or (b) R 3a is H or C1-C 12 alkyl, and R 3b together with the carbon atom to which it is attached, the adjacent R 3b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 4a and R 4b is, in each occurrence, independently: (a) H or C-C 12 alkyl; or (b) R 4a is H or C1-C 12 alkyl, and R 4b together with the carbon atom to which it is attached, the adjacent R 4b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 5 and R 6 are each independently H or methyl; R 7 is H or C1-C 20 is alkyl; R 8 is OH, -N(R 9 )(C=O)R 10 , -(C=O)NR 9 R 10 , -NR 9 R 10 , -(C=O)OR 11 or -O(C=O)R 11 where R 8 Ga-NR 9 R 10 When G 3 is a C4-C6 alkylene; R 9 and R 10 are each independently H or C-C 12 is alkyl; R 11 is aralkyl; a, b, c, and d are each independently an integer from 1 to 24; x is 0, 1, or 2; wherein each alkyl, alkylene and aralkyl may be optionally substituted. or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0317] In some embodiments, L 1 and L 2 each independently represents -O(C=O)-, -(C=O)O- or a direct bond. 1 and G 2 Each is independently —(C═O)— or a direct bond. 1 and L 2 are each independently —O(C═O)—, —(C═O)O—, or a direct bond; G 1 and G 2 is each independently —(C═O)— or a direct bond.

[0318] In several different embodiments, L 1 and L 2 are each independently -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a -, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a , -OC(=O)NR a -, -NR a C(=O)O-, -NR a S(O) x NR a -, -NR a S(O) x -or-S(O) x NR a -It is.

[0319] In other embodiments of the above embodiments, the compound has the following formula (VIA) or formula (VIB): [ka] It has one of the following.

[0320] In some embodiments, the compound has the formula (VIA): In other embodiments, the compound has the formula (VIB):

[0321] In any of the preceding embodiments, L 1 or L 2 One of the groups is —O(C═O)—. For example, in some embodiments, L 1 and L 2 Each of is —O(C═O)—.

[0322] In some different embodiments of any of the foregoing, L 1 or L 2 One of the groups is —(C═O)O—. For example, in some embodiments, L 1 and L 2 Each of is —(C═O)O—.

[0323] In a different embodiment, L 1 or L 2 As used herein, a "direct bond" refers to a bond that is bonded to a group (e.g., L 1 or L 2 ) is absent. For example, in some embodiments, L 1 and L 2 Each of is a direct bond.

[0324] In another different embodiment of the above, R 1a and R 1b For at least one case of R 1a is H or C1-C 12 alkyl, and R 1b together with the carbon atom to which it is attached, the adjacent R 1band together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0325] In yet another different embodiment, R 4a and R 4b For at least one case of R 4a is H or C1-C 12 alkyl, and R 4b together with the carbon atom to which it is attached, the adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0326] In a further embodiment, R 2a and R 2b For at least one case of R 2a is H or C1-C 12 alkyl, and R 2b together with the carbon atom to which it is attached, the adjacent R 2b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0327] In any other different embodiment of the above, R 3a and R 3b For at least one case of R 3a is H or C1-C 12 alkyl, and R 3b together with the carbon atom to which it is attached, the adjacent R 3b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0328] A "carbon-carbon" double bond has the following structure: [ka] [During the ceremony, R c and R d is, in each occurrence, independently H or a substituent. For example, in some embodiments, R cand R d is, in each occurrence independently, H, C-C 12 Alkyl or cycloalkyl, e.g., H or C1-C 12 It is alkyl.

[0329] In various other embodiments, the compound has the following formula (VIC) or (VID): [ka] wherein e, f, g, and h each independently represent an integer of 1 to 12. It has one of the following.

[0330] In some embodiments, the compound has the formula (VIC): In other embodiments, the compound has the formula (VID).

[0331] In various embodiments of compounds of Formula (VIC) or (VID), e, f, g, and h are each independently an integer from 4 to 10.

[0332] In another different embodiment, [ka] or both, independently, have one of the following structures: [ka]

[0333] In certain of the above embodiments, a, b, c, and d are each independently an integer of 2 to 12 or an integer of 4 to 12. In other embodiments, a, b, c, and d are each independently an integer of 8 to 12 or an integer of 5 to 9. In some specific embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In further embodiments, a is 3. In still other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In further embodiments, a is 7. In still other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In further embodiments, a is 11. In still other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In further embodiments, a is 15. In still other embodiments, a is 16.

[0334] In some embodiments, b is 1. In other embodiments, b is 2. In further embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In further embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In further embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In further embodiments, b is 15. In yet other embodiments, b is 16.

[0335] In some embodiments, c is 1. In other embodiments, c is 2. In further embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In further embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In further embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In further embodiments, c is 15. In yet other embodiments, c is 16.

[0336] In some particular embodiments, d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In further embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In further embodiments, d is 7. In still other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In further embodiments, d is 11. In still other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In further embodiments, d is 15. In still other embodiments, d is 16.

[0337] In some embodiments, e is 1. In other embodiments, e is 2. In further embodiments, e is 3. In yet other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In further embodiments, e is 7. In still other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In further embodiments, e is 11. In still other embodiments, e is 12.

[0338] In some embodiments, f is 1. In other embodiments, f is 2. In further embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In further embodiments, f is 7. In still other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In further embodiments, f is 11. In still other embodiments, f is 12.

[0339] In some embodiments, g is 1. In other embodiments, g is 2. In further embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In further embodiments, g is 7. In still other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In further embodiments, g is 11. In still other embodiments, g is 12.

[0340] In some embodiments, h is 1. In other embodiments, e is 2. In further embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In further embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In further embodiments, h is 11. In yet other embodiments, h is 12.

[0341] In some other various embodiments, a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.

[0342] The sum of a and b and c and d are factors that can be varied to obtain lipids with desired properties. In one embodiment, a and b are selected so that their sum is an integer ranging from 14 to 24. In another embodiment, c and d are selected so that their sum is an integer ranging from 14 to 24. In further embodiments, the sum of a and b and the sum of c and d are the same. For example, in some embodiments, the sum of a and b and the sum of c and d are both the same integer, which can range from 14 to 24. In further embodiments, a, b, c, and d are selected so that the sum of a and b and the sum of c and d are 12 or greater.

[0343] R 1a , R 2a , R 3a and R 4a The substituents of R are not particularly limited. 1a , R 2a , R 3a and R 4a At least one of R is H. In certain embodiments, 1a , R 2a , R 3a and R 4a is H in each occurrence. In certain other embodiments, R 1a , R 2a , R 3a and R 4a At least one of the following is C1-C 12 In certain other embodiments, R 1a , R 2a , R 3a and R 4a At least one of R is C-C alkyl. 1a , R 2a , R 3a and R 4aAt least one of is C1-C6 alkyl. In some of the above embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0344] In certain of the above embodiments, R 1a , R 1b , R 4a and R 4b In each case, C1-C 12 It is alkyl.

[0345] In further embodiments of the above, R 1b , R 2b , R 3b and R 4b At least one of is H or R 1b , R 2b , R 3b and R 4b is H in each case.

[0346] In certain of the above embodiments, R 1b together with the carbon atom to which it is attached, the adjacent R 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond. 4b together with the carbon atom to which it is attached, the adjacent R 4b and together with the carbon atom to which it is attached form a carbon-carbon double bond.

[0347] In the above embodiment, R 5 and R 6 The substituents of R are not particularly limited. 5 or R 6 One of R is methyl. 5 or R 6 Each of is methyl.

[0348] In the above embodiment, R 7The substituents of R are not particularly limited. 7 is C6-C 16 In some other embodiments, R 7 is C6-C9 alkyl. In some of these embodiments, R 7 is -(C=O)OR b , -O(C=O)R b , -C(=O)R b , -OR b , -S(O) x R b , -S-SR b , -C(=O)SR b , -SC(=O)R b , -NR a R b , -NR a C(=O)R b , -C(=O)NR a R b , -NR a C(=O)NR a R b , -OC(=O)NR a R b , -NR a C(=O)OR b , -NR a S(O) x NR a R b , -NR a S(O) x R b or -S(O) x NR a R b wherein R a is H or C1-C 12 alkyl; b is C1-C 15 alkyl; and x is 0, 1, or 2. For example, in some embodiments, R 7 is -(C=O)OR b or -O(C=O)R b is replaced by

[0349] In various embodiments of the above embodiments, R b is branch C3-C 15For example, in some embodiments, R b has one of the following structures: [ka]

[0350] In certain embodiments, R 8 is OH.

[0351] In other embodiments, R 8 -N(R 9 )(C=O)R 10 In some other embodiments, R 8 -(C=O)NR 9 R 10 In a further embodiment, R 8 Ha-NR 9 R 10 In some of the above embodiments, R 9 and R 10 is each independently H or C1-C8 alkyl, e.g., H or C1-C3 alkyl. In more specific embodiments of these embodiments, the C1-C8 alkyl or C1-C3 alkyl is unsubstituted or substituted with hydroxyl. In other embodiments of these embodiments, R 9 and R 10 are each methyl.

[0352] In a further embodiment, R 8 -(C=O)OR 11 In some of these embodiments, R 11 is benzyl.

[0353] In an even more specific embodiment, R 8 has one of the following structures: [ka]

[0354] In still other embodiments of the compounds, G3 is C2-C5 alkylene, e.g., C2-C4 alkylene, C3 alkylene, or C4 alkylene. In some of these embodiments, R 8 is OH. In other embodiments, G 2 does not exist, and R 7 is C1-C2 alkylene, for example, methyl.

[0355] In various different embodiments, the compound has one of the structures shown in Table 5 below. [Table 25] [Table 26] [Table 27] [Table 28] [Table 29] [Table 30]

[0356] In one embodiment, the cationic lipid has the following formula (VII): [ka] [During the ceremony, X and X′ are each independently N or CR; Y and Y′ are each independently absent, —O(C═O)—, —(C═O)O—, or NR, with the proviso that: a) when X is N, Y does not exist; b) when X' is N, Y' is absent; c) when X is CR, Y is —O(C═O)—, —(C═O)O—, or NR; d) when X' is CR, Y' is -O(C=O)-, -(C=O)O- or NR; L 1 and L 1 Each ' is independently -O(C=O)R 1 , -(C=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) z R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c or -NR a C(=O)OR 1 and; L 2 and L 2 Each ' is independently -O(C=O)R 2 , -(C=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) z R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f ;-NR d C(=O)OR 2 or R 2 is a direct bond to; G 1 , G 1 ', G 2 and G2 ' are each independently C2-C 12 Alkylene or C2-C 12 alkenylene; G 3 is C2-C 24 Heteroalkylene or C2-C 24 heteroalkenylene; R a , R b , R d and R e is, in each occurrence independently, H, C-C 12 Alkyl or C2-C 12 is alkenyl; R c and R f are, in each instance independently, C1-C 12 Alkyl or C2-C 12 is alkenyl; R, at each occurrence, is independently H or C-C 12 is alkyl; R 1 and R 2 is, in each occurrence independently, a branched C-C 24 Alkyl or branched C6-C 24 is alkenyl; z is 0, 1, or 2; wherein, unless otherwise specified, each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene, and heteroalkenylene is independently substituted or unsubstituted. or a pharmaceutically acceptable salt or stereoisomer thereof.

[0357] In another different embodiment of formula (VII): X and X′ are each independently N or CR; Y and Y′ are each independently absent or NR, with the proviso that: a) when X is N, Y does not exist; b) when X' is N, Y' is absent; c) when X is CR, Y is NR; d) when X' is CR, Y' is NR; L 1 and L 1 Each ' is independently -O(C=O)R 1 , -(C=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) z R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c or -NR a C(=O)OR 1 and; L 2 and L 2 Each ' is independently -O(C=O)R 2 , -(C=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) z R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f ;-NR d C(=O)OR 2 or R 2 is a direct bond to; G 1 , G 1 ', G 2 and G 2' are each independently C2-C 12 Alkylene or C2-C 12 alkenylene; G 3 is C2-C 24 Alkylene oxide or C2-C 24 alkenylene oxide; R a , R b , R d and R e is, in each occurrence, independently: H, C-C 12 Alkyl or C2-C 12 is alkenyl; R c and R f are, in each instance independently, C1-C 12 Alkyl or C2-C 12 is alkenyl; R, at each occurrence, is independently H or C-C 12 is alkyl; R 1 and R 2 is, in each occurrence independently, a branched C-C 24 Alkyl or branched C6-C 24 is alkenyl; z is 0, 1, or 2; Herein, unless otherwise specified, each alkyl, alkenyl, alkylene, alkenylene, alkylene oxide and alkenylene oxide is independently substituted or unsubstituted.

[0358] In some embodiments, G 3 is C2-C 24 Alkylene oxide or C2-C 24 In certain embodiments, G is an alkenylene oxide. 3 is unsubstituted. 3 is substituted, e.g., substituted with hydroxyl. In a more specific embodiment, G 3 is C2-C 12 alkylene oxide, e.g., in some embodiments, G 3is a C3-C7 alkylene oxide, or in other embodiments, G 3 is C3-C 12 It is an alkylene oxide.

[0359] In other embodiments, G 3 is C2-C 24 Alkyleneaminyl or C2-C 24 Alkenyleneaminyl, for example, C-C 12 In some of these embodiments, G is alkyleneaminyl. 3 is unsubstituted. In other of these embodiments, G 3 is substituted with C1-C6 alkyl.

[0360] In some embodiments, X and X' are each N and Y and Y' are absent. In other embodiments, X and X' are each CR and Y and Y' are each NR. In some of these embodiments, R is H.

[0361] In certain embodiments, each of X and X', and each of CR and Y and Y', independently, is -O(C=O)- or -(C=O)O-.

[0362] In some of the above embodiments, the compound has the following formula (VIIA), (VIIB), (VIIC), (VIID), (VIIE), (VIIF), (VIIG), or (VIIH): [ka] [ka] [In the formula, R d is, at each occurrence, independently H or optionally substituted C1-C6 alkyl. For example, in some embodiments, R d is H. In other embodiments, R dis C1-C6 alkyl, e.g., methyl. In other embodiments, R d is a substituted C1-C6 alkyl, e.g., C1-C6 alkyl substituted with -O(C=O)R, -(C=O)OR, -NRC(=O)R, or -C(=O)N(R), wherein R, in each occurrence, is independently H or C1-C 12 It is alkyl.

[0363] In some of the foregoing embodiments, L 1 and L 1 Each ' is independently -O(C=O)R 1 , -(C=O)OR 1 or -C(=O)NR b R c and L 2 and L 2 Each ' is independently -O(C=O)R 2 , -(C=O)OR 2 or -C(=O)NR e R f For example, in some embodiments, L 1 and L 1 ' are -(C=O)OR 1 and L 2 and L 2 ' are -(C=O)OR 2 In another embodiment, L 1 and L 1 ' are -(C=O)OR 1 and L 2 and L 2 ' are -C(=O)NR e R f In another embodiment, L 1 and L 1 ' are -C(=O)NR b R c and L 2 and L 2 ' are -C(=O)NR e R f is.

[0364] In some of the foregoing embodiments, G1 , G 1 ', G 2 and G 2 Each ' is independently C2-C8 alkylene, for example, C4-C8 alkylene.

[0365] In some of the above embodiments, R 1 or R 2 each, in each occurrence, independently, a branched C-C 24 For example, in some embodiments, R 1 and R 2 is, in each occurrence, independently, the structure: [ka] [During the ceremony, R 7a and R 7b is, in each occurrence, independently H or C-C 12 is alkyl; a is an integer ranging from 2 to 12; Here, R 7a , R 7b and a are R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or from 8 to 12.

[0366] In some of the above embodiments, R 7a is H. For example, in some embodiments, R 7a is H in each occurrence. In other different embodiments of the above, R 7b is C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0367] In a different embodiment, R1 or R 2 , or both, in each instance independently, have the structure: [ka]

[0368] In some of the above embodiments, R b , R c , R e and R f are, when present, each independently C3-C 12 For example, in some embodiments, R b , R c , R e and R f When present, R is n-hexyl; in other embodiments, R b , R c , R e and R f When present, is n-octyl.

[0369] In various different embodiments, the compound has one of the structures shown in Table 6 below. [Table 31] [Table 32]

[0370] In one embodiment, the cationic lipid has the following formula (VIII): [ka] [During the ceremony, X is N and Y is absent; or X is CR and Y is NR; L 1 is -O(C=O)R 1 , -(C=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c or -NR a C(=O)OR 1 and; L 2 is -O(C=O)R 2 , -(C=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f ;-NR d C(=O)OR 2 or R 2 is a direct bond to; L 3 is -O(C=O)R 3 or -(C=O)OR 3 and; G 1 and G 2 are each independently C2-C 12 Alkylene or C2-C 12 alkenylene; G 3 is C1-C 24 Alkylene, C2-C 24 Alkenylene, C1-C 24 Heteroalkylene or C2-C24 heteroalkenylene; R a , R b , R d and R e are each independently H or C-C 12 Alkyl or C1-C 12 is alkenyl; R c and R f are each independently C1-C 12 Alkyl or C2-C 12 is alkenyl; Each R is independently H or C1-C 12 is alkyl; R 1 , R 2 and R 3 are each independently C1-C 24 Alkyl or C2-C 24 is alkenyl; x is 0, 1, or 2; wherein, unless otherwise specified, each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene, and heteroalkenylene is independently substituted or unsubstituted. or a pharmaceutically acceptable salt or stereoisomer thereof.

[0371] In a further embodiment of Formula (VIII): X is N and Y is absent; or X is CR and Y is NR; L 1 is -O(C=O)R 1 , -(C=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR aC(=O)NR b R c , -OC(=O)NR b R c or -NR a C(=O)OR 1 and L 2 is -O(C=O)R 2 , -(C=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f ;-NR d C(=O)OR 2 or R 2 is a direct bond to; L 3 is -O(C=O)R 3 or -(C=O)OR 3 and; G 1 and G 2 are each independently C2-C 12 Alkylene or C2-C 12 alkenylene; When X is CR and Y is NR, G 3 is C1-C 24 Alkylene, C2-C 24 Alkenylene, C1-C 24 Heteroalkylene or C2-C 24 heteroalkenylene; when X is N and Y is absent, G 3 is C1-C 24 Heteroalkylene or C2-C 24 heteroalkenylene; R a , R b , Rd and R e are each independently H or C-C 12 Alkyl or C1-C 12 is alkenyl; R c and R f are each independently C1-C 12 Alkyl or C2-C 12 is alkenyl; Each R is independently H or C1-C 12 is alkyl; R 1 , R 2 and R 3 are each independently C1-C 24 Alkyl or C2-C 24 is alkenyl; x is 0, 1, or 2; Herein, unless otherwise specified, each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene and heteroalkenylene is independently substituted or unsubstituted.

[0372] In other embodiments of Formula (VIII): X is N and Y is absent, or X is CR and Y is NR; L 1 is -O(C=O)R 1 , -(C=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c or -NR a C(=O)OR 1 and; L 2 is -O(C=O)R 2 , -(C=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f ;-NR d C(=O)OR 2 or R 2 is a direct bond to; L 3 is -O(C=O)R 3 or -(C=O)OR 3 and; G 1 and G 2 are each independently C2-C 12 Alkylene or C2-C 12 alkenylene; G 3 is C1-C 24 Alkylene, C2-C 24 Alkenylene, C1-C 24 Heteroalkylene or C2-C 24 heteroalkenylene; R a , R b , R d and R e are each independently H or C-C 12 Alkyl or C1-C 12 is alkenyl; R c and R f are each independently C1-C 12 Alkyl or C2-C 12 is alkenyl; Each R is independently H or C1-C 12 is alkyl; R 1 , R 2 and R 3 each independently represents a branched C6-C 24 Alkyl or branched C6-C 24 is alkenyl; x is 0, 1, or 2; Herein, unless otherwise specified, each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene and heteroalkenylene is independently substituted or unsubstituted.

[0373] In certain embodiments, G 3 is unsubstituted. In a more specific embodiment, G 3 is C2-C 12 alkylene, for example, in some embodiments, G 3 is C3-C7 alkylene; or in other embodiments, G 3 is C3-C 12 In some embodiments, G is alkylene. 3 is a C2 or C3 alkylene.

[0374] In other embodiments, G 3 is C1-C 12 Heteroalkylene, e.g., C-C 12 It is an aminyl alkylene.

[0375] In certain embodiments, X is N and Y is absent. In other embodiments, X is CR and Y is NR, for example, in some of these embodiments, R is H.

[0376] In some of the above embodiments, the compound has one of the following formulas (VIIIA), (VIIIB), (VIIIC), or (VIIID): [ka]

[0377] In some of the foregoing embodiments, L 1 is -O(C=O)R 1 , -(C=O)OR 1 or -C(=O)NR b R and L 2 is -O(C=O)R 2 , -(C=O)OR 2 or -C(=O)NR e R f In another specific embodiment, L 1 -(C=O)OR 1 and L 2 -(C=O)OR 2 In any of the above embodiments, L 3 -(C=O)OR 3 is.

[0378] In some of the above embodiments, G 1 and G 2 are each independently C2-C 12 Alkylene, e.g., C4-C 10 It is alkylene.

[0379] In some of the above embodiments, R 1 , R 2 and R 3 each independently represents a branched C6-C 24 For example, in some embodiments, R 1 , R 2 and R 3 each independently have the structure: [ka] [During the ceremony R 7a and R 7b is, in each occurrence, independently H or C-C 12 is alkyl; a is an integer ranging from 2 to 12; where R 7a , R 7b and a is R 1 and R 2are each selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or from 8 to 12.

[0380] In some of the above embodiments, R 7a is H. For example, in some embodiments, R 7a is H in each occurrence. In other different embodiments of the above, at least one R 7b is C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0381] In some of the foregoing embodiments, X is CR, Y is NR, and R 3 is C1-C such as ethyl, propyl or butyl 12 In some of these embodiments, R 1 and R 2 each independently represents a branched C6-C 24 It is alkyl.

[0382] In a different embodiment, R 1 , R 2 and R 3 each independently has the structure: [ka]

[0383] In certain embodiments, R 1 and R 2 and R 3 each independently represents a branched C6-C 24 alkyl, and R 3 is C1-C 24 Alkyl or C2-C 24 It is alkenyl.

[0384] In some of the above embodiments, R b , R c , R e and R f are each independently C3-C 12 For example, in some embodiments, R b , R c , R e and R f is n-hexyl, and in other embodiments, R b , R c , R e and R f is n-octyl.

[0385] In various different embodiments, the compound has one of the structures shown in Table 7 below. [Table 33] [Table 34]

[0386] In one embodiment, the cationic lipid has the following formula (IX): [ka] [During the ceremony, L 1 is -O(C=O)R 1 , -(C=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R cor -NR a C(=O)OR 1 and; L 2 is -O(C=O)R 2 , -(C=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f and; -NR d C(=O)OR 2 or R 2 is a direct bond to; G 1 and G 2 are each independently C2-C 12 Alkylene or C2-C 12 alkenylene; G 3 is C1-C 24 Alkylene, C2-C 24 alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene; R a , R b , R d and R e are each independently H or C-C 12 Alkyl or C1-C 12 is alkenyl; R c and R f are each independently C1-C 12 Alkyl or C2-C 12 is alkenyl; R 1 and R 2 each independently represents a branched C6-C24 Alkyl or branched C6-C 24 is alkenyl; R 3 -N(R 4 )R 5 and; R 4 is C1-C 12 is alkyl; R 5 is a substitution C1-C 12 is alkyl; x is 0, 1, or 2; wherein, unless otherwise specified, each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl, and aralkyl is independently substituted or unsubstituted. or a pharmaceutically acceptable salt or stereoisomer thereof.

[0387] In certain embodiments, G 3 is unsubstituted. In a more specific embodiment, G 3 is C2-C 12 Alkylene, for example, in some embodiments, G 3 is C3-C7 alkylene; or in other embodiments, G 3 is C3-C 12 In some embodiments, G is alkylene. 3 is a C2 or C3 alkylene.

[0388] In some of the above embodiments, the compound has the following formula (IXA): [ka] [During the ceremony, y and z are each independently an integer ranging from 2 to 12, e.g., from 2 to 6, from 4 to 10, or e.g., 4 or 5. In certain embodiments, y and z are the same and are selected from 4, 5, 6, 7, 8 and 9.

[0389] In some of the foregoing embodiments, L1 is -O(C=O)R 1 , -(C=O)OR 1 or -C(=O)NR b R c and L 2 is -O(C=O)R 2 , -(C=O)OR 2 or -C(=O)NR e R f For example, in some embodiments, L 1 and L 2 are -(C=O)OR 1 and -(C=O)OR 2 In another embodiment, L 1 -(C=O)OR 1 and L 2 is -C(=O)NR e R f In another embodiment, L 1 is -C(=O)NR b R c and L 2 is -C(=O)NR e R f is.

[0390] In other of the foregoing embodiments, the compound has one of formula (IXB), formula (IXC), formula (IXD) or formula (IXE). [ka]

[0391] In some of the above embodiments, the compound has formula (IXB), in other embodiments, the compound has formula (IXC), and in still other embodiments, the compound has formula (IXD). In other embodiments, the compound has formula (IXE).

[0392] In several different embodiments of the above, the compound has the following formula (IXF), formula (IXG), formula (IXH), or formula (IXJ): [ka] wherein y and z are each independently an integer ranging from 2 to 12, for example, from 2 to 6, for example, 4.

[0393] In some of the above embodiments, y and z are each independently an integer ranging from 2 to 10, from 2 to 8, from 4 to 10, or from 4 to 7. For example, in some embodiments, y is 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, z is 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, y and z are the same, while in other embodiments, y and z are different.

[0394] In some of the above embodiments, R 1 or R 2 , or both are branched C6-C 24 For example, in some embodiments, R 1 and R 2 each independently have the structure: [ka] [During the ceremony, R 7a and R 7b is, in each occurrence, independently H or C-C 12 is alkyl; a is an integer ranging from 2 to 12; where R 7a , R 7b and a are R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or from 8 to 12.

[0395] In some of the above embodiments, R 7a is H. For example, in some embodiments, R 7ais H in each occurrence. In other different embodiments of the above, R 7b is C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0396] In a different embodiment, R 1 or R 2 , or both, have one of the following structures: [ka]

[0397] In some of the above embodiments, R b , R c , R e and R f are each independently C3-C 12 For example, in some embodiments, R b , R c , R e and R f is n-hexyl, and in other embodiments, R b , R c , R e and R f is n-octyl.

[0398] In any of the preceding embodiments, R 4 is substituted or unsubstituted: methyl, ethyl, propyl, n-butyl, n-hexyl, n-octyl, or n-nonyl. For example, in some embodiments, R 4 is unsubstituted. In other embodiments, R 4 -OR g , -NR g C(=O)R h , -C(=O)NR g R h , -C(=O)R h , -OC(=O)R h , -C(=O)OR hAND -OR i OH, wherein: R g is, at each occurrence, independently H or C1-C6 alkyl; R h is, independently at each occurrence, C1-C6 alkyl; R i is, independently at each occurrence, C1-C6 alkylene.

[0399] In other embodiments of the above embodiments, R 5 is substituted: methyl, ethyl, propyl, n-butyl, n-hexyl, n-octyl, or n-nonyl. 5 is substituted ethyl or substituted propyl. In another different embodiment, R 5 is substituted hydroxyl. In a further embodiment, R 5 -OR g , -NR g C(=O)R h , -C(=O)NR g R h , -C(=O)R h , -OC(=O)R h , -C(=O)OR h AND -OR i OH, wherein: R g is, at each occurrence, independently H or C1-C6 alkyl; R h is, independently at each occurrence, C1-C6 alkyl; R i is, independently at each occurrence, C1-C6 alkylene.

[0400] In other embodiments, R 4 is unsubstituted methyl, and R 5 is substituted: methyl, ethyl, propyl, n-butyl, n-hexyl, n-octyl, or n-nonyl. In some of these embodiments, R5 is substituted with hydroxyl.

[0401] In some other specific embodiments, R 3 has the following structure: [ka]

[0402] In various different embodiments, the compound has one of the structures shown in Table 8 below. [Table 35] [Table 36] [Table 37]

[0403] In one embodiment, the cationic lipid has the following formula (X): [ka] [During the ceremony, G 1 -OH, -NR 3 R 4 , -(C=O)NR 5 or -NR 3 (C=O)R 5 and; G 2 is -CH2- or -(C=O)-; R, at each occurrence, is independently H or OH; R 1 and R 2 are each independently a branched saturated or unsaturated C 12 -C 36 is alkyl; R 3 and R 4 are each independently H or a straight chain or branched saturated or unsaturated C1-C6 alkyl; R5 is a linear or branched saturated or unsaturated C1-C6 alkyl; n is an integer ranging from 2 to 6. or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0404] In some embodiments, R 1 and R 2 are each independently a branched saturated or unsaturated C 12 -C 30 Alkyl, C 12 -C 20 Alkyl or C 15 -C 20 In some specific embodiments, R 1 and R 2 Each is saturated. In certain embodiments, R 1 and R 2 At least one of the is unsaturated.

[0405] In some of the above embodiments, R 1 and R 2 has the following structure: [ka]

[0406] In some of the above embodiments, the compound has the following formula (XA): [ka] [During the ceremony, R 6 and R 7 is, in each occurrence, independently H or a straight-chain or branched, saturated or unsaturated C-C 14 is alkyl; a and b are each independently an integer ranging from 1 to 15; However, R 6 and a and R 7 and b are each independently R 1 and R 2each independently represents a branched saturated or unsaturated C 12 -C 36 alkyl]

[0407] In some of the above embodiments, the compound has the following formula (XB): [ka] [During the ceremony, R 8 , R 9 , R 10 and R 11 are each independently a straight-chain or branched saturated or unsaturated C4-C 12 alkyl, provided that R 8 and R 9 and R 10 and R 11 are each independently R 1 and R 2 each independently a branched saturated or unsaturated C 12 -C 36 alkyl] In some embodiments of Formula (XB), R 8 , R 9 , R 10 and R 11 are each independently a straight-chain or branched, saturated or unsaturated C-C 10 In certain embodiments of formula (XB), R 8 , R 9 , R 10 and R 11 At least one of R is unsaturated. 8 , R 9 , R 10 and R 11 Each of the is saturated.

[0408] In some of the above embodiments, the compound has the formula (XA), and in other embodiments, the compound has the formula (XB).

[0409] In some of the above embodiments, G 1 is —OH, and in some embodiments, G 1 Ha-NR 3 R 4 For example, in some embodiments, G 1 is -NH, -NHCH, or -N(CH). In certain embodiments, G 1 -(C=O)NR 5 In certain other embodiments, G 1 Ha-NR 3 (C=O)R 5 For example, in some embodiments, G 1 is -NH(C=O)CH3 or -NH(C=O)CH2CH2CH3.

[0410] In some of the above embodiments, G 2 is -CH2-. In some different embodiments, G 2 is -(C=O)-.

[0411] In some of the above embodiments, n is an integer ranging from 2 to 6, for example, in some embodiments, n is 2, 3, 4, 5, or 6. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.

[0412] In certain embodiments of the above embodiments, R 1 , R 2 , R 3 , R 4 and R 5 At least one of R is unsubstituted. For example, in some embodiments, 1 , R 2 , R 3 , R 4 and R 5 are each unsubstituted. In some embodiments, R 3 is substituted. In other embodiments, R 4 is substituted. In a further embodiment, R 5is substituted. In certain specific embodiments, R 3 and R 4 Each of R is substituted. 3 , R 4 or R 5 The above substituent is hydroxyl. In certain embodiments, R 3 and R 4 are each substituted with a hydroxyl.

[0413] In some of the above embodiments, at least one R is OH. In other embodiments, each R is H.

[0414] In various different embodiments, the compound has one of the structures shown in Table 9 below. [Table 38] [Table 39] [Table 40]

[0415] In one embodiment, the cationic lipid has the following formula (XI): [ka] [During the ceremony, L 1 is -O(C=O)R 1 , -(C=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NRb R c , -OC(=O)NR b R c or -NR a C(=O)OR 1 and; L 2 is -O(C=O)R 2 , -(C=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR c C(=O)NR e R f , -OC(=O)NR e R f ;-NR d C(=O)OR 2 or R 2 is a direct bond to; G 1a and G 2a are each independently C2-C 12 Alkylene or C2-C 12 alkenylene; G 3 is C1-C 24 Alkylene, C2-C 24 alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene; R a , R b , R d and R e are each independently H or C-C 12 Alkyl or C2-C 12 is alkenyl; R c and R f are each independently C1-C 12 Alkyl or C2-C 12 is alkenyl; R 1 and R 2 each independently represents a branched C6-C 24 Alkyl or branched C6-C 24 is alkenyl; R 3a is -C(=O)N(R 4a )R 5a or -C(=O)OR 6 and; R 4a is C1-C 12 is alkyl; R 5a is H or C1-C8 alkyl or C2-C8 alkenyl; R 6 is H, aryl or aralkyl; x is 0, 1, or 2; wherein each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl, and aralkyl is independently substituted or unsubstituted. or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0416] In certain embodiments of formula (XI), G 3 is unsubstituted. In more specific embodiments of formula (XI), G 3 is C3-C 12 In some embodiments of Formula (XI), G is alkylene. 3 is a C2 or C3 alkylene.

[0417] In some of the above embodiments of formula (XI), the compound has the following structure (IA): [ka] [wherein y1 and z1 are each independently an integer in the range of 2 to 12, for example, an integer of 2 to 6, for example, 4].

[0418] In some of the above embodiments of Formula (XI), L 1 is -O(C=O)R1 , -(C=O)OR 1 or -C(=O)NR b R c and L 2 is -O(C=O)R 2 , -(C=O)OR 2 or -C(=O)NR e R f For example, in some embodiments of formula (XI), L 1 and L 2 are -(C=O)OR 1 and -(C=O)OR 2 In another embodiment of formula (XI), L 1 -(C=O)OR 1 and L 2 is -C(=O)NR e R f In another embodiment of formula (XI), L 1 is -C(=O)NR b R c and L 2 is -C(=O)NR e R f is.

[0419] In other of the foregoing embodiments, the compound has one of the following formulas (IB), (IC), (ID), or (IE): [ka]

[0420] In some of the above embodiments, the compound has formula (XIB), in other embodiments, the compound has formula (XIC), and in still other embodiments, the compound has formula (XID). In other embodiments, the compound has formula (XIE).

[0421] In several different embodiments described above, the compound has one of the following formulas (XIF), (XIG), (XIH), or (XIJ): [ka] [wherein y1 and z1 are each independently an integer in the range of 2 to 12, for example, an integer of 2 to 6, for example, 4].

[0422] In some of the above embodiments of Formula (XI), y1 and z1 are each independently an integer ranging from 2 to 10, from 2 to 8, from 4 to 10, or from 4 to 7. For example, in some embodiments of Formula (XI), y1 is 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments of Formula (XI), z1 is 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments of Formula (XI), y1 and z1 are the same; in other embodiments of Formula (XI), y1 and z1 are different.

[0423] In some of the above embodiments of formula (XI), R 1 or R 2 , or both of these are branched C6-C 24 For example, in some embodiments of formula (XI), R 1 and R 2 each independently have the structure: [ka] [During the ceremony, R 7a and R 7b is, in each occurrence, independently H or C-C 12 is alkyl; a is an integer ranging from 2 to 12; Here, R 7a , R 7b and a are R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or from 8 to 12.

[0424] In some of the above embodiments of formula (XI), R 7ais H. For example, in some embodiments of formula (XI), R 7a is H in each occurrence. In other different embodiments of the above, R 7b is C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0425] In different embodiments of formula (XI), R 1 or R 2 or both of which have the following structure: [ka]

[0426] In some of the above embodiments of formula (XI), R b , R c , R e and R f are each independently C3-C 12 For example, in some embodiments of formula (XI), R b , R c , R e and R f is n-hexyl, and in other embodiments of formula (XI), R b , R c , R e and R f is n-octyl.

[0427] In some of the above embodiments of formula (XI), R 3a is -C(=O)N(R 4a )R 5a In more specific embodiments of formula (XI), R 4a is ethyl, propyl, n-butyl, n-hexyl, n-octyl, or n-nonyl. In certain embodiments of formula (XI), R 5ais H, methyl, ethyl, propyl, n-butyl, n-hexyl, or n-octyl. In some of these embodiments of formula (XI), R 4a and / or R 5a may be optionally substituted with a substituent, for example, hydroxyl.

[0428] In some embodiments of Formula (XI), R 3a is -C(=O)OR 6 In certain embodiments of formula (XI), R 6 is benzyl, and in other embodiments, R 6 is H.

[0429] In some of the above embodiments of formula (XI), R 4a , R 5a and R 6 are independently -OR g , -NR g C(=O)R h , -C(=O)NR g R h , -C(=O)R h , -OC(=O)R h , -C(=O)OR h AND -OR i Optionally substituted with one or more substituents selected from the group consisting of OH, wherein: R g is, at each occurrence, independently H or C1-C6 alkyl; R h is, independently in each occurrence, C1-C6 alkyl; R i is, independently in each occurrence, C1-C6 alkylene.

[0430] In specific embodiments of formula (XI), R 3a has one of the following structures: [ka]

[0431] In various different embodiments, the compound has one of the structures shown in Table 10 below. [Table 41] [Table 42] [Table 43]

[0432] In another embodiment, the cationic lipid has the following formula (XII): [ka] [During the ceremony, L 1 is -O(C=O)R 1 , -(C=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c or -NR a C(=O)OR 1 and; L 2 is -O(C=O)R 2 , -(C=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R2 , -C(=O)NR e R f , -NR c C(=O)NR e R f , -OC(=O)NR e R f ;-NR d C(=O)OR 2 or is a direct bond; G 1b and G 2b are each independently C1-C 12 Alkylene or C2-C 12 alkenylene; G 3 is C1-C 24 Alkylene, C2-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a , R b , R d and R e are each independently H or C-C 12 Alkyl or C2-C 12 is alkenyl; R c and R f are each independently C1-C 12 Alkyl or C2-C 12 is alkenyl; R 1 and R 2 each independently represents a branched C6-C 24 Alkyl or branched C6-C 24 is alkenyl; R 3b Ha-NR 4b C(=O)R 5b and; R 4b is H, C1-C 12 Alkyl or C2-C 12 is alkenyl; R 4b When is H, R 5b is C2-C 12 Alkyl or C2-C 12 alkenyl; or R4b is C1-C 12 Alkyl or C2-C 12 When R is alkenyl, 5 is C1-C 12 Alkyl or C2-C 12 is alkenyl; x is 0, 1, or 2; wherein each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently substituted or unsubstituted. or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0433] In certain embodiments of Formula (XII), G 3 is unsubstituted. In more specific embodiments of formula (XII), G 3 is C1-C 12 Alkylene, e.g., G 3 is C3-C5 alkylene or G 3 is C3-C 12 It is alkylene.

[0434] In some of the above embodiments, the cationic lipid has the following formula (XIIA): [ka] (wherein y2 and z2 each independently represent an integer ranging from 1 to 12). or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0435] In some of the above embodiments of formula (XIIA), L 1 and L 2 are each independently -O(C=O)R 1 or -(C=O)OR 1 is.

[0436] In other of the above embodiments, the compound has one of the following formulae (XIIB) or (XIIC): [ka]

[0437] In some of the above embodiments, the compound has formula (XIIB), and in other embodiments, the compound has formula (XIIC).

[0438] In some embodiments, the compound has one of the following formulas (XIID) or (XIIE): [ka] (wherein y2 and z2 each independently represent an integer ranging from 1 to 12).

[0439] In some of the foregoing embodiments of Formula (XII), y2 and z2 are each independently an integer ranging from 2 to 12, e.g., from 2 to 10, from 2 to 8, from 4 to 7, or from 4 to 10. For example, in some embodiments of Structure (II), y2 is 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments of Formula (XII), z2 is 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments of Formula (XII), y2 and z2 are the same; in other embodiments of Formula (XII), y2 and z2 are different.

[0440] In some of the above embodiments of formula (XII), R 1 or R 2 , or both of these are branched C6-C 24 For example, in some embodiments of Formula (XII), R 1 and R 2 each independently have the structure: [ka] [During the ceremony, R 7a and R 7b is, in each occurrence, independently H or C-C 12 alkyl; a is an integer ranging from 2 to 12; Here, R 7a , R 7b and a are R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or from 8 to 12.

[0441] In some of the above embodiments of formula (XII), R 7a is H. For example, in some embodiments of Formula (XII), R 7a is H in each occurrence. In other different embodiments of the above, R 7b is C1-C8 alkyl. For example, in some embodiments of Formula (XII), C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0442] In different embodiments of formula (XII), R 1 or R 2 , or both of them have one of the following structures: [ka]

[0443] In some of the above embodiments of formula (XII), R 4b is H, methyl, ethyl, propyl, or octyl. In some embodiments of Formula (XII), R 5b is methyl, ethyl, propyl, heptyl or octyl, for example n-heptyl or n-octyl.

[0444] In certain related embodiments of Formula (XII), R 4b and R 5b are independently -OR g , -NR g C(=O)R h, -C(=O)NR g R h , -C(=O)R h , -OC(=O)R h , -C(=O)OR h AND -OR h OH, wherein R g is, in each occurrence, independently H or C1-C6 alkyl; R h is, independently in each occurrence, C1-C6 alkyl; R i is, independently in each occurrence, C1-C6 alkylene.

[0445] In certain specific embodiments of formula (XII), R 3b has one of the following structures: [ka]

[0446] In various different embodiments, the compound of formula (XII) has one of the structures shown in Table 11 below. [Table 44] [Table 45] [Table 46] [Table 47]

[0447] In one embodiment, the cationic lipid has the following structure: [ka] [During the ceremony, R 1is optionally substituted C1-C 24 Alkyl or optionally substituted C-C 24 is alkenyl; R 2 and R 3 are each independently an optionally substituted C-C 36 is alkyl; R 4 and R 5 are each independently an optionally substituted C1-C6 alkyl, or R 4 and R 5 are joined together with the N atom to which they are attached to form a heterocyclyl or heteroaryl; L 1 , L 2 and L 3 are each independently an optionally substituted C-C 18 is alkylene; G 1 is a direct bond, -(CH2) n O(C=O)-, -(CH2) n (C=O)O- or -(C=O)-; G 2 and G 3 are each independently —(C═O)O— or —O(C═O)—; n is an integer greater than 0. or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0448] In some embodiments, the compound has the structure: [ka]

[0449] In some embodiments, the compound has the structure: [ka]

[0450] In some embodiments, R 1 is optionally substituted C6-C 18 Alkyl or C 14 -C 18 In certain embodiments, R 1 is C8 alkyl, C9 alkyl, C 10 Alkyl, C 12 Alkyl, C 14 Alkyl or C 16 In some more specific embodiments, R 1 is C 16 In certain more specific embodiments, R 1 is unbranched. In some embodiments, R 1 is branched. In certain embodiments, R 1 is non-substituted.

[0451] In some embodiments, G 1 is a direct bond, -(CH2) n O(C=O)- or -(CH2) n (C=O)O-. In certain embodiments, G 1 is a direct bond. In some more specific embodiments, G 1 Ha-(CH2) n (C=O)O-, and n is greater than 1. In some embodiments, n is 1-20. In some embodiments, n is 1 to 10. In some embodiments, n is 5 to 11. In some embodiments, n is 6 to 10. In certain more specific embodiments, n is 5, 6, 7, 8, 9, or 10. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In certain embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

[0452] In some embodiments, L 1is C1-C6 alkylene. In certain embodiments, L 1 is a C2 alkylene, a C3 alkylene, or a C4 alkylene. 1 is unbranched. In certain more specific embodiments, L 1 is non-substituted.

[0453] In some embodiments, R 2 is C8-C 24 In some embodiments, R 3 is C8-C 24 In some more specific embodiments, R 2 and R 3 Both, C8-C 24 In some embodiments, R 2 and R 3 are each independently 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl, C 15 Alkyl, C 16 Alkyl, C 18 Alkyl or C 20 In certain embodiments, R 2 is branched. In a more specific embodiment, R 3 is branched. In some more specific embodiments, R 2 and R 3 each independently has one of the following structures: [ka] [In the formula, R 6 and R 7 are each independently C2-C 12 alkyl]

[0454] In some embodiments, R 2 and R 3 each independently has one of the following structures: [ka]

[0455] In some embodiments, L 2 and L 3 are each independently C4-C 10 In certain embodiments, L 2 and L 3 and are both C5 alkylene. In some more specific embodiments, L 2 and L 3 and are both C alkylene. In certain embodiments, L 2 and L 3 and are both C alkylene. In some more specific embodiments, L 2 and L 3 and are both C alkylene. In some embodiments, L 2 is unbranched. In some embodiments, L 3 In a more specific embodiment, L 2 is unsubstituted. In some embodiments, L 2 is non-substituted.

[0456] In some embodiments, R 4 and R 5 is each independently C1-C6 alkyl. In a more specific embodiment, R 4 and R 5 and R are both methyl. 4 and R 5 and R are both ethyl. 4 is methyl and R 5 is n-butyl. In some embodiments, R 4 and R 5 and R are both n-butyl. 4 is methyl and R 5 is n-hexyl.

[0457] In some embodiments, R4 and R 5 are joined together with the N to which they are attached to form a heterocyclyl. In certain embodiments, the heterocyclyl is a 5-membered heterocyclyl. In some embodiments, the heterocyclyl has the structure: [ka]

[0458] In various different embodiments, the compound has one of the structures shown in Table 12 below. [Table 48] [Table 49] [Table 50] [Table 51] [Table 52] [Table 53] [Table 54]

[0459] In one embodiment, the lipid compound has the following structure: [ka] [During the ceremony, R2 and R3 are independently H, C1- 14 Alkyl, C2- 14 Alkenyl, -R * selected from the group consisting of YR″ and YR″; R4 is a C3-6 carbocyclic ring, -(CH2) n Q, -(CH2) nand N(R)R, wherein each n is independently selected from 1, 2, 3, 4, and 5; R8 is selected from the group consisting of C3-6 carbocycles and heterocycles; each R is independently selected from the group consisting of C alkyl, C alkenyl, and H; Each R' is independently C1- 18 Alkyl, C2- 18 Alkenyl, -R * selected from the group consisting of YR″, -YR″ and H; Each R'' is independently C3- 14 Alkyl and C3- 14 alkenyl; Each R * are independent, C1- 12 Alkyl and C2- 12 alkenyl; each Y is independently selected from C3-6 carbocycle; l is selected from 1, 2, 3, 4 and 5; m is selected from 5, 6, 7, 8 and 9; M1 is a bond of M'; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group. or a salt or isomer thereof.

[0460] In certain embodiments, the lipid compound has the structure: [ka]

[0461] In certain embodiments, the lipid compound has the formula: [ka]

[0462] In one embodiment, the lipid compound has the formula: [ka]

[0463] In another embodiment, the lipid compound has the formula: [ka]

[0464] In certain embodiments, the lipid compound has the formula: [ka]

[0465] In certain embodiments, the lipid compound is a compound in which R4 is —(CH2) n Q, -(CH2) n and -CQ(R)2, wherein said Q is -N(R)R8.

[0466] In some embodiments, M and M' are independently -C(O)O- or -OC(O)-.

[0467] In other embodiments, R4 is selected from any of the following groups: [ka]

[0468] In other embodiments, R4 is selected from any of the following groups: [ka]

[0469] In other embodiments, the cationic lipid is a lipid disclosed in WO 2020 / 0061367, the entire contents of which are incorporated herein by reference. For example, in some embodiments of the present invention, the cationic lipid described herein has the formula [ka] [During the ceremony, R 1 is C5-30 alkyl, C5-20 alkenyl, -R * selected from the group consisting of -YR'', -YR'', and -R'M'R'; R 2 and R 3 are independently H, C alkyl, C alkenyl, -R * YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is selected from the group consisting of hydrogen, C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -(CH2)oC(R10)2(CH2)n-oQ, -CHQR, -CQ(R)2, -C(O)NQR and unsubstituted Ci-e alkyl, wherein said Q is carbocycle, heterocycle, -OR, -O(CH2)nN(R)2, - C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S( 0)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -N(R)S(0)2R8, -O(CH2)nOR, -N(R) C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(O R)S(0)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2 , -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, -(CH2)nN(R)2 and -C(R)N(R)2C(O)OR, wherein each 0 is independently selected from 1, 2, 3 and 4, and each n is independently selected from 1, 2, 3, 4 and 5; each R5 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M'' is a bond, C1-13 alkyl, or C2-13 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycles and heterocycles; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; R10 is selected from the group consisting of H, OH, C1-3 alkyl, and C2-3 alkenyl; Each R is independently C1-6 alkyl, C1-3 alkyl-aryl, C2-3 alkenyl, (CH2)qOR * and H, each q is independently selected from 1, 2, and 3; Each R' is independently C alkyl, C alkenyl, -R * selected from the group consisting of YR″, -YR″ and H; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R * is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and when R is —(CH)Q, —(CH)CHQR, —CHQR, or —CQ(R), then (i) when n is 1, 2, 3, 4, or 5, Q is not —N(R), or (ii) when n is 1 or 2, Q is not a 5-, 6-, or 7-membered heterocycloalkyl. or an N-oxide thereof or a salt or isomer thereof.

[0470] Other cationic lipids include those of formula (III): [ka] [During the ceremony, R1 is C5-30 alkyl, C5-20 alkenyl, -R * selected from the group consisting of -YR'', -YR'', and -R'M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R * YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is selected from the group consisting of hydrogen, C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -(CH2)oC(R10)2(CH2)n-oQ, -CHQR, -CQ(R)2, -C(O)NQR and unsubstituted Ci-e alkyl, wherein Q is carbocycle, heterocycle, -OR, -O(CH2)nN(R)2, -C (O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O )2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -N(R)S(O)2R8, -O(CH2)nOR, -N(R)C (=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR )S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, selected from -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, -(CH2)nN(R)2 and -C(R)N(R)2C(O)OR, wherein each 0 is independently selected from 1, 2, 3 and 4, and each n is independently selected from 1, 2, 3, 4 and 5; Rx is selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, -(CH2)vOH and -(CH2)VN(R)2; where v is selected from 1, 2, 3, 4, 5 and 6; each R5 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; and M'' is a bond, C1-13 alkyl, or C2-i3 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycles and heterocycles; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; R10 is selected from the group consisting of H, OH, C1-3 alkyl, and C2-3 alkenyl; Each R is independently C1-6 alkyl, C1-3 alkyl-aryl, C2-3 alkenyl, (CH2)qOR * and H, each q is independently selected from 1, 2, and 3; Each R' is independently C alkyl, C alkenyl, -R * selected from the group consisting of YR″, -YR″ and H; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R * is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. or an N-oxide thereof, or a salt or isomer thereof or a salt or isomer thereof.

[0471] In another embodiment, the present invention relates to compounds of formula (I) wherein R4 is selected from the group consisting of -(CH2)nQ, -(CH2)nCHQR, -(CH2)oC(R12)2(CH2)n-oQ, -CHQR, -CQ(R)2 and -C(O)NQR, wherein said Q is -(CH2)nN(R)2.

[0472] In another embodiment, the present invention relates to compounds of formula (III) wherein R4 is selected from the group consisting of -(CH2)nQ, -(CH2)nCHQR, -(CH2)oC(R12)2(CH2)n-oQ, -CHQR, -CQ(R)2 and -C(O)NQR, wherein said Q is -(CH2)nN(R)2.

[0473] In some embodiments, some compounds of Formula (I) include those in which, when R4 is -(CH2)nQ, -(CH2)nCHQR, -CHQR, or -CQ(R)2, (i) when n is 1, 2, 3, 4, or 5, Q is not -N(R)2, or (ii) when n is 1 or 2, Q is not a 5-, 6-, or 7-membered heterocycloalkyl.

[0474] For example, when R4 is -(CH2)nQ, -(CH2)nCHQR, -(CH2)oC(R10)2(CH2)n-oQ, -CHQR, or -CQ(R)2, (i) when n is 1, 2, 3, 4, or 5, Q is not -N(R)2, or (ii) when n is 1 or 2, Q is not a 5-, 6-, or 7-membered heterocycloalkyl.

[0475] In another embodiment, another portion of the compounds of formula (I) is R1 is C5-30 alkyl, C5-20 alkenyl, -R * selected from the group consisting of -YR'', -YR'', and -R'M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R * YR'', -YR'', and -R *OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is selected from the group consisting of hydrogen, C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -(CH2)oC(R10)2(CH2)n-oQ, -CHQR, -CQ(R)2, -C(O)NQR and unsubstituted Ci-e alkyl, wherein Q is C3-6 carbocycle, 5-14 membered heteroaryl having one or more heteroatoms selected from N, O and S, -OR, -O(CH2)nN(R )2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, - N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -N(R)S(O)2R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR 9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(O R)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, -(C H2)nN(R)2 and 5-14 membered heterocycloalkyl having one or more heteroatoms selected from N, O, and S, which are substituted with one or more substituents selected from oxo (=O), OH, amino, mono- or dialkylamino, and Ci-3 alkyl, wherein each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M'' is a bond, C1-13 alkyl, or C2-13 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycles and heterocycles; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; R10 is selected from the group consisting of H, OH, C1-3 alkyl, and C2-3 alkenyl; Each R is independently C1-6 alkyl, C1-3 alkyl-aryl, C2-3 alkenyl, (CH2)qOR * and H; Each R' is independently selected from Ci-ib alkyl, C2-18 alkenyl, -R * selected from the group consisting of YR″, —YR″ and H; each q is independently selected from 1, 2, and 3; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R * is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The present invention includes compounds or N-oxides or salts or isomers thereof, which are

[0476] In yet another embodiment, another portion of the compounds of Formula (I) is R1 is C5-30 alkyl, C5-20 alkenyl, -R * selected from the group consisting of -YR'', -YR'', and -R'M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R * YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is selected from the group consisting of hydrogen, C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -(CH2)oC(R10)2(CH2)n-oQ, -CHQR, -CQ(R)2, -C(O)NQR and unsubstituted Ci-e alkyl, wherein Q is C3-6 carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O and S, -OR, -O(CH2)nN(R)2, -C(O)O R, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R) 2, -CRN(R)2C(O)OR, -N(R)R8, -N(R)S(O)2R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N (R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR) Selected from C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, -(CH2)nN(R)2 and -C(=NR9)N(R)2, where each 0 is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5; Q is a 5-14 membered heterocycle, and when (i) R4 is -(CH2)nQ where n is 1 or 2, or (ii) R4 is -(CH2)nCHQR1 where n is 1, or (iii) R4 is -CHQR and -CQ(R)2, then Q is a 5-14 membered heteroaryl or an 8-14 membered heterocycloalkyl; each R5 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M'' is a bond, C1-13 alkyl, or C2-i3 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycles and heterocycles; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; R10 is selected from the group consisting of H, OH, C1-3 alkyl, and C2-3 alkenyl; Each R is independently C1-6 alkyl, C1-3 alkyl-aryl, C2-3 alkenyl, (CH2)qOR * and H, each q is independently selected from 1, 2, and 3; Each R' is independently C alkyl, C alkenyl, -R * selected from the group consisting of YR″, -YR″ and H; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R * is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The present invention includes compounds or their N-oxides or salts or isomers.

[0477] In yet another embodiment, another portion of the compounds of formula (I) is R1 is C5-30 alkyl, C5-20 alkenyl, -R * selected from the group consisting of -YR'', -YR'', and -R'M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R * YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is selected from the group consisting of hydrogen, C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -(CH2)oC(R10)2(CH2)n-oQ, -CHQR, -CQ(R)2, -C(O)NQR and unsubstituted Ci-e alkyl, wherein Q is C3-6 carbocycle, 5-14 membered heteroaryl having one or more heteroatoms selected from N, O and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N (R)R8, -N(R)S(O)2R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N( R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR )C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, -(CH2)nN(R)2, each 0 is independently selected from 1, 2, 3 and 4 and -C(=NR9)N(R)2, and each n is independently selected from 1, 2, 3, 4 and 5; each R5 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M'' is a bond, C1-13 alkyl, or C2-13 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycles and heterocycles; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; R10 is selected from the group consisting of H, OH, C1-3 alkyl, and C2-3 alkenyl, and each R is independently C1-6 alkyl, C1-3 alkyl-aryl, C2-3 alkenyl, (CH2)qOR * and H, each q is independently selected from 1, 2, and 3; Each R' is independently C alkyl, C alkenyl, -R * selected from the group consisting of YR″, -YR″ and H; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R * is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The present invention includes compounds or N-oxides or salts or isomers thereof, which are

[0478] In yet another embodiment, another portion of the compounds of formula (I) is R1 is C5-30 alkyl, C5-20 alkenyl, -R * selected from the group consisting of -YR'', -YR'', and -R''M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R * YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is selected from the group consisting of hydrogen, C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -(CH2)oC(R10)2(CH2)n-oQ, -CHQR, -CQ(R)2, -C(O)NQR and unsubstituted Ci-e alkyl, wherein said Q is carbocycle, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -N(R)S(O)2R8, -O(CH2)nOR, -N(R)C(=NR9 )N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S( O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N -C(R)N(R)R, -C(R)N(R)R, -C(O)N(R)R, -C(O)N(R)R, -(CH)N(R)R and -C(R)N(R)R; each 0 is independently selected from 1, 2, 3 and 4, and each n is independently selected from 1, 2, 3, 4 and 5; each R5 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M'' is a bond, C1-13 alkyl, or C2-13 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycles and heterocycles; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; R10 is selected from the group consisting of H, OH, C1-3 alkyl, and C2-3 alkenyl; each R is independently selected from the group consisting of C alkyl, C alkyl-aryl, C alkenyl, and H; Each R' is independently C alkyl, C alkenyl, -R * YR'', -YR'', (CH2)qOR * and H, q is independently selected from 1, 2, and 3; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R * is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13; This includes compounds that are

[0479] In yet another embodiment, another portion of the compounds of Formula (I) is R1 is C5-30 alkyl, C5-20 alkenyl, -R * selected from the group consisting of -YR'', -YR'', and -R''M'R'; R2 and R3 are independently H, C2-14 alkyl, C2-14 alkenyl, -R * YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is -(CH2)nQ or -(CH2)nCHQR, where Q is -N(R)2 and n is selected from 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M' is a bond, C1-13 alkyl, or C2-13 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R is independently selected from the group consisting of C alkyl, C alkyl-aryl, C alkenyl, and H; Each R' is independently C alkyl, C alkenyl, -R * selected from the group consisting of YR″, -YR″ and H; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R * is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The present invention includes compounds or N-oxides or salts or isomers thereof, which are

[0480] In yet another embodiment, another portion of the compounds of formula (I) is R1 is C5-30 alkyl, C5-20 alkenyl, -R *selected from the group consisting of -YR'', -YR'', and -R''M'R'; R2 and R3 are independently C1-14 alkyl, C2-14 alkenyl, -R * YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is selected from the group consisting of -(CH2)nQ, -(CH2)nCHQR, -CHQR, and -CQ(R)2, where Q is -N(R)2 and n is selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M'' is a bond, C1-13 alkyl, or C2-13 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R is independently selected from the group consisting of C alkyl, C alkyl-aryl, C alkenyl, and H; Each R' is independently C alkyl, C alkenyl, -R * selected from the group consisting of YR″, -YR″ and H; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R * is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The present invention includes compounds or N-oxides or salts or isomers thereof, which are

[0481] In yet another embodiment, another portion of the compounds of formula (I) is R1 is C5-30 alkyl, C5-20 alkenyl, -R * selected from the group consisting of -YR'', -YR'', and -R''M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R * YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is -C(O)NQR, wherein Q is selected from carbocycle, heterocycle, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -(CH2)nN(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR and -C(R)N(R)2C(O)OR, where each n is independently selected from 1, 2, 3, 4 and 5; each R5 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M'' is a bond, C1-13 alkyl, or C2-13 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of Ci-6 alkyl, C1-3 alkyl-aryl, C2-3 alkenyl, and H; Each R' is independently C alkyl, C alkenyl, -R * YR'', -YR'', (CH2)qOR * and H, where each q is independently selected from 1, 2, and 3; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R * is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; This includes compounds that are

[0482] In some embodiments, some compounds of Formula (III) include compounds in which, when R4 is -(CH2)nQ, -(CH2)nCHQR, -CHQR, or -CQ(R)2, (i) when n is 1, 2, 3, 4, or 5, Q is not -N(R)2, or (ii) when n is 1 or 2, Q is not a 5-, 6-, or 7-membered heterocycloalkyl.

[0483] In another embodiment, another portion of the compounds of formula (III) is R1 is C5-30 alkyl, C5-20 alkenyl, -R * selected from the group consisting of -YR'', -YR'', and -R''M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R * YR'', -YR'', and -R *OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is selected from the group consisting of hydrogen, C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -(CH2)oC(R10)2(CH2)n-oQ, -CHQR, -CQ(R)2, -C(O)NQR and unsubstituted Ci-e alkyl, wherein Q is C3-6 carbocycle, 5-14 membered heteroaryl having one or more heteroatoms selected from N, O and S, -OR, -O(CH2)nN(R) 2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N (R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -N(R)S(O)2R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9 )N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(O R)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, -(C H2)nN(R)2 and 5-14 membered heterocycloalkyl having one or more heteroatoms selected from N, O and S, which are substituted with one or more substituents selected from oxo (=O), OH, amino, mono- or dialkylamino and C1-3 alkyl, wherein each 0 is independently selected from 1, 2, 3 and 4, and each n is independently selected from 1, 2, 3, 4 and 5; Rx is selected from the group consisting of Ci-6 alkyl, C2-6 alkenyl, -(CfkXOH, and -(CH2)VN(R)2; where v is selected from 1, 2, 3, 4, 5 and 6; each R5 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M'' is a bond, C1-13 alkyl, or C2-13 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycles and heterocycles; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; R10 is selected from the group consisting of H, OH, C1-3 alkyl, and C2-3 alkenyl; Each R is independently C1-6 alkyl, C1-3 alkyl-aryl, C2-3 alkenyl, (CH2)qOR * and H; Each R' is independently C alkyl, C alkenyl, -R * selected from the group consisting of YR″, —YR″ and H; each q is independently selected from 1, 2, and 3; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R * is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The present invention includes compounds or N-oxides or salts or isomers thereof, which are

[0484] In yet another embodiment, another portion of the compounds of Formula (III) is R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR'', -YR'', and -R'M'R'; R2 and R3 are independently selected from the group consisting of H, Ci-i4 alkyl, C2-14 alkenyl, -R*YR'', -YR'' and -R*OR'', or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is selected from the group consisting of hydrogen, C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -(CH2)oC(R12)2(CH2)n-oQ, -CHQR, -CQ(R)2, -C(O)NQR and unsubstituted Ci-e alkyl, wherein Q is C3-6 carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O and S, -OR, -O(CH2)nN(R)2, -C(O)OR , -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R) 2, -CRN(R)2C(O)OR, -N(R)R8, -N(R)S(O)2R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N (R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR )C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, -(CH2)nN(R)2 and -C(=NR9)N(R)2, each with 0 is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5; Q is a 5-14 membered heterocycle, and when (i) R4 is —(CH2)nQ where n is 1 or 2, or (ii) R4 is —(CH2)nCHQR where n is 1, or (iii) R4 is —CHQR and —CQ(R)2, then Q is a 5-14 membered heteroaryl or an 8-14 membered heterocycloalkyl; Rx is selected from the group consisting of Ci-6 alkyl, C2-6 alkenyl, -(CH2)vOH and -(CH2)VN(R)2; wherein v is selected from 1, 2, 3, 4, 5, and 6; each R5 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M'' is a bond, C1-13 alkyl, or C2-i3 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycles and heterocycles; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; R12 is selected from the group consisting of H, OH, C1-3 alkyl, and C2-3 alkenyl; Each R is independently C1-6 alkyl, C1-3 alkyl-aryl, C2-3 alkenyl, (CH2)qOR * and H, each q is independently selected from 1, 2, and 3; Each R' is independently selected from Ci-ib alkyl, C2-18 alkenyl, -R * selected from the group consisting of YR″, -YR″ and H; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R * is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The present invention includes compounds or N-oxides or salts or isomers thereof, which are

[0485] In yet another embodiment, another portion of the compounds of formula (III) is R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR'', -YR'', and -R''M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R * YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is selected from the group consisting of hydrogen, C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -(CH2)oC(R12)2(CH2)n-oQ, -CHQR, -CQ(R)2, -C(O)NQR and unsubstituted Ci-e alkyl, wherein Q is a C3-6 carbocycle, a 5-14 membered heterocycle having one or more heteroatoms selected from N, O and S; Teroaryl, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R) C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -N(R)S(O )2R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, - N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=N -R9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, -(CH2)nN(R)2, where each 0 is independently selected from 1, 2, 3, and 4, -C(=NR9)N(R)2, where each 0 is independently selected from 1, 2, 3, and 4, and where each n is independently selected from 1, 2, 3, 4, and 5; Rx is selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, -(CH2)vOH and -(CH2)VN(R)2; wherein v is selected from the group consisting of 1, 2, 3, 4, 5, and 6; each R5 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M'' is a bond, C1-13 alkyl, or C2-13 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycles and heterocycles; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; R12 is selected from the group consisting of H, OH, C1-3 alkyl, and C2-3 alkenyl; Each R is independently C1-6 alkyl, C1-3 alkyl-aryl, C2-3 alkenyl, (CH2)qOR * and H, Each q is independently Ci-is alkyl, C2-18 alkenyl, -R * selected from the group consisting of YR″, -YR″ and H; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R * is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The present invention includes compounds or N-oxides or salts or isomers thereof, which are

[0486] In yet another embodiment, another portion of the compounds of formula (III) is R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR'', -YR'', and -R''M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R * YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is selected from the group consisting of hydrogen, C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -(CH2)oC(R12)2(CH2)n-oQ, -CHQR, -CQ(R)2, -C(O)NQR and unsubstituted Ci-e alkyl, wherein said Q is carbocycle, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -N(R)S(O)2R8, -O(CH2)nOR, -N(R)C(=NR9) selected from N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, -(CH2)nN(R)2 and -C(R)N(R)2C(O)OR, wherein each o is independently selected from 1, 2, 3 and 4, and each n is independently selected from 1, 2, 3, 4 and 5; Rx is selected from the group consisting of Ci-6 alkyl, C2-6 alkenyl, -(CH2)vOH and -(CH2)VN(R)2; where v is selected from 1, 2, 3, 4, 5 and 6; each R5 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M'' is a bond, C1-13 alkyl, or C2-i3 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycles and heterocycles; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; R12 is selected from the group consisting of H, OH, C1-3 alkyl, and C2-3 alkenyl; each R is independently selected from the group consisting of C alkyl, C alkyl-aryl, C alkenyl, and H; each R' is independently Ci-ib alkyl, C2-ie alkenyl, -R * YR'', -YR'', (CH2)qOR * and H, each q is independently selected from 1, 2, and 3; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R *is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; This includes compounds that are

[0487] In yet another embodiment, another portion of the compounds of Formula (III) is R1 is C5-30 alkyl, C5-20 alkenyl, -R * selected from the group consisting of -YR'', -YR'', and -R''M'R'; R2 and R3 are independently H, C2-14 alkyl, C2-14 alkenyl, -R * YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is -(CH2)nQ or -(CH2)nCHQR, where Q is -N(R)2 and n is selected from 3, 4, and 5; Rx is selected from the group consisting of Ci-6 alkyl, C2-6 alkenyl, -(CH2)vOH, and -(CH2)VN(R)2; where v is selected from 1, 2, 3, 4, 5 and 6; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M'' is a bond, C1-13 alkyl, or C2-i3 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R is independently selected from the group consisting of C alkyl, C alkyl-aryl, C alkenyl, and H; Each R' is independently selected from C1-is alkyl, C2-i8 alkenyl, -R * selected from the group consisting of YR″, -YR″ and H; each R″ is independently selected from the group consisting of C3-15 alkyl and C3-1 alkenyl; Each R * is independently selected from the group consisting of Ci-i2 alkyl and Ci-i2 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; The present invention includes compounds or N-oxides or salts or isomers thereof, which are

[0488] In yet another embodiment, another portion of the compounds of formula (III) is R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR'', -YR'', and -R''M'R'; R2 and R3 are independently C1-14 alkyl, C2-14 alkenyl, -R * YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is selected from the group consisting of -(CH2)nQ, -(CH2)nCHQR, -CHQR, and -CQ(R)2, where Q is -N(R)2 and n is selected from 1, 2, 3, 4, and 5; Rx is selected from the group consisting of Ci-6 alkyl, C2-6 alkenyl, -(CH2)vOH and -(CH2)VN(R)2; where v is selected from 1, 2, 3, 4, 5 and 6; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M'' is a bond, C1-13 alkyl, or C2-13 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R is independently selected from the group consisting of C alkyl, C alkyl-aryl, C alkenyl, and H; Each R' is independently C alkyl, C alkenyl, -R * selected from the group consisting of YR″, -YR″ and H; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R * is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; In certain embodiments, some of the compounds of formula (I) are of formula (IA): [ka] wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; Mi is a bond or M'; R4 is hydrogen, unsubstituted C1-3 alkyl, -(CH2)oC(R12)2(CH2)n-oQ, -C(O)NQR, or -(CH2)nQ, and Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2, -NHC(=CHR9 )N(R), -OC(O)N(R), -N(R)C(O)OR, -(CH)N(R), heteroaryl, or heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R), -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R and R are independently selected from the group consisting of H, C alkyl, and C alkenyl. or an N-oxide, or a salt or isomer thereof. For example, m is 5, 7, or 9. For example, Q is OH, —NHC(S)N(R)2, or —NHC(O)N(R)2. For example, Q is —N(R)C(O)R, or —N(R)S(O)2R.

[0489] In certain embodiments, some of the compounds of Formula (I) have the formula (IB): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein all variables are as defined herein. For example, m is selected from 5, 6, 7, 8, and 9; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl. For example, m is 5, 7, or 9. In certain embodiments, some compounds of Formula (I) are those of Formula (II): [ka] [During the ceremony, l is selected from 1, 2, 3, 4 and 5; M is a bond or M'; R is hydrogen, unsubstituted C alkyl, -(CH)OC(R)(CH)Q, -C(O)NQR or -(CH)Q, where n is 2, 3 or 4 and Q is OH, -NHC(S)N(R), -NHC(O)N(R), -N(R)C(O)R, -N(R)S(O)R, -N(R)R, -NHC(=NR)N(R), -NHC(=CHR)N(R), -OC(O)N(R), -N(R)C(O)OR, -(CH)N(R), heteroaryl, or heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R and R are independently selected from the group consisting of H, CI-M alkyl, and C-M alkenyl. or an N-oxide thereof or a salt or isomer thereof.

[0490] In certain embodiments, some of the compounds of Formula (I) are represented by Formula (IIa), Formula (IIb), Formula (IIc), or Formula (IIe): [ka] wherein R4 is as defined herein. or an N-oxide thereof or a salt or isomer thereof.

[0491] In certain embodiments, some of the compounds of Formula (I) are represented by Formula (IId): [ka] [During the ceremony, n is 2, 3, or 4; m, M, M'', R', R'', and R2-R6 are as described herein. or an N-oxide thereof, or a salt or isomer thereof. For example, each of R2 and R3 can be selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.

[0492] In another embodiment, some of the compounds of formula (I) have the formula (IIf): [ka] [During the ceremony, n is 2, 3, or 4; m, M, M'', R', R'', and R2-R6 are as described herein. or an N-oxide thereof, or a salt or isomer thereof. For example, each of R2 and R3 can be independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl, and n is selected from 2, 3, and 4.

[0493] In another embodiment, some of the compounds of formula (I) have the formula (IIg): [ka] wherein l, m, M, M1, R', R2, and R3 are as described herein. or an N-oxide thereof, or a salt or isomer thereof. For example, each of R2 and R3 can be independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl, wherein l is selected from 1, 2, 3, 4, and 5, and m is selected from 5, 6, 7, 8, and 9.

[0494] Another aspect of the present invention is a compound of formula (VI): [ka] [During the ceremony, R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR'', -YR'', and -R'M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R *YR'', -YR'', and -R * OR″, or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; each R5 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, and M'' is a bond, C1-13 alkyl, or C2-13 alkenyl; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R is independently selected from the group consisting of H, C alkyl, and C alkenyl; R N is H, or Ci-3 alkyl; Each R' is independently C alkyl, C alkenyl, -R * selected from the group consisting of YR″, -YR″ and H; each R" is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; Each R * is independently selected from the group consisting of C alkyl and C alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; Xa and Xb are each independently O or S; R10 is H, halo, -OH, R, -N(R)2, -CN, -N3, -C(O)OH, -C(O)OR, -OC(O)R, -OR, -SR, -S(O)R, -S(O)OR, -S(O)2 OR, -NO2, -S(O)2N(R)2, -N(R)S(O)2R, -NH(CH2)tiN(R)2, -NH(CH2)PiO(CH2)qiN(R)2, -NH(CH2)SIOR, -N((CH2)SIOR)2, -N(R)-carbocycle, -N(R)-heterocycle, -N(R)-aryl, -N(R)-heteroaryl, -N(R)(CH2)ti-carbocycle, -N(R)(CH2)ti-heterocycle, -N(R)(CH2)ti-aryl, -N(R)(CH2)u-heteroaryl, carbocycle, heterocycle, aryl and heteroaryl; m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13; n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; r is 0 or 1; t1 is selected from 1, 2, 3, 4 and 5; p1 is selected from 1, 2, 3, 4 and 5; q1 is selected from 1, 2, 3, 4 and 5; s1 is selected from 1, 2, 3, 4 and 5. or an N-oxide thereof or a salt or isomer thereof.

[0495] In some embodiments, a portion of the compound of Formula (VI) is represented by Formula (VI-a): [ka] [During the ceremony, R1a and R1b are independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; R2 and R3 are independently C1-14 alkyl, C2-14 alkenyl, -R * YR'', -YR'', and -R * OR'', or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle. or an N-oxide or salt or isomer thereof.

[0496] In other embodiments, some of the compounds of Formula (VI) are represented by Formula (VII): [ka] [During the ceremony, l is selected from 1, 2, 3, 4 and 5; Mi is a bond or M'; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl. or an N-oxide or salt or isomer thereof.

[0497] In other embodiments, some of the compounds of Formula (VI) are represented by Formula (VIII): [ka] [During the ceremony, l is selected from 1, 2, 3, 4 and 5; Mi is a bond or M'; Ra and Rb are independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; R2 and R3 are independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl. or an N-oxide thereof or a salt or isomer thereof.

[0498] The compounds of any of formula (I), formula (IA), formula (VI), formula (VI-a), formula (VII) or formula (VIII), when applicable, include one or more of the following features:

[0499] In some embodiments, Mi is M'.

[0500] In some embodiments, M and M' are independently -C(O)O- or -OC(O)-.

[0501] In some embodiments, at least one of M and M' is -C(O)O- or -OC(O)-.

[0502] In certain embodiments, at least one of M and M' is -OC(O)-.

[0503] In certain embodiments, M is -OC(O)- and M' is -C(O)O-. In some embodiments, M is -C(O)O- and M' is -OC(O)-. In certain embodiments, M and M' are each -OC(O)-. In some embodiments, M and M' are each -C(O)O-.

[0504] In certain embodiments, at least one of M and M' is -OC(O)-M''-C(O)O-.

[0505] In some embodiments, M and M' are independently -SS-.

[0506] In some embodiments, at least one of M and M' is -SS.

[0507] In some embodiments, one of M and M' is -C(O)O- or -OC(O)- and the other is -SS-. For example, M is -C(O)O- or -OC(O)- and M' is -SS-, or M' is -C(O)O- or -OC(O)- and M is -SS-.

[0508] In some embodiments, one of M and M' is -OC(O)-M"-C(O)O-, and M" is a bond, a Ci-i3 alkyl, or a C2-13 alkenyl. In other embodiments, M" is a C1-6 alkyl or a C2-6 alkenyl. In certain embodiments, M" is a C1-4 alkyl or a C2-4 alkenyl. For example, in some embodiments, M" is a Ci alkyl. For example, in some embodiments, M" is a C2 alkyl. For example, in some embodiments, M" is a C3 alkyl. For example, in some embodiments, M" is a C4 alkyl. For example, in some embodiments, M" is a C2 alkenyl. For example, in some embodiments, M" is a C3 alkenyl. For example, in some embodiments, M" is a C4 alkenyl.

[0509] In some embodiments, l is 1, 3, or 5.

[0510] In some embodiments, R4 is hydrogen.

[0511] In some embodiments, R4 is not hydrogen.

[0512] In some embodiments, R4 is unsubstituted methyl or -(CH2)nQ, where Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, or -N(R)S(O)2R.

[0513] In some embodiments, Q is OH.

[0514] In some embodiments, Q is —NHC(S)N(R) 2 .

[0515] In some embodiments, Q is —NHC(O)N(R) 2 .

[0516] In some embodiments, Q is —N(R)C(O)R.

[0517] In some embodiments, Q is —N(R)S(O)R.

[0518] In some embodiments, Q is —O(CH 2 ) n N(R) 2 .

[0519] In some embodiments, Q is —O(CH 2 ) n OR.

[0520] In some embodiments, Q is —N(R)R 8 .

[0521] In some embodiments, Q is -NHC(=NR9)N(R)2.

[0522] In some embodiments, Q is -NHC(=CHR9)N(R)2.

[0523] In some embodiments, Q is —OC(O)N(R) 2 .

[0524] In some embodiments, Q is —N(R)C(O)OR.

[0525] In some embodiments, n is 2.

[0526] In some embodiments, n is 3.

[0527] In some embodiments, n is 4.

[0528] In some embodiments, Mi is absent.

[0529] In some embodiments, at least one R5 is hydroxyl, e.g., one R5 is hydroxyl.

[0530] In some embodiments, at least one R6 is hydroxyl, e.g., one R6 is hydroxyl.

[0531] In some embodiments, one of R5 and R6 is hydroxyl.For example, one R5 is hydroxyl and each R6 is hydrogen.For example, one R6 is hydroxyl and each R5 is hydrogen.

[0532] In some embodiments, Rx is Ci-6 alkyl. In some embodiments, Rx is Ci-3 alkyl. For example, Rx is methyl. For example, Rx is ethyl. For example, Rx is propyl.

[0533] In some embodiments, Rx is -(CFkXOFl and v is 1, 2, or 3. For example, Rx is methanoyl. For example, Rx is ethanoyl. For example, Rx is propanoyl.

[0534] In some embodiments, Rx is -(CH2)vN(R)2, where v is 1, 2, or 3, and each R is H or methyl. For example, Rx is methaneamino, methylmethaneamino, or dimethylmethaneamino. For example, Rx is aminomethanyl, methylaminomethanyl, or dimethylaminomethanyl. For example, Rx is aminoethanyl, methylaminoethanyl, or dimethylaminoethanyl. For example, Rx is aminopropanyl, methylaminopropanyl, or dimethylaminopropanyl.

[0535] In some embodiments, R' is Ci-ib alkyl, C2-18 alkenyl, -R * YR'', or -YR''.

[0536] In some embodiments, R2 and R3 are independently C3-14 alkyl or C3-14 alkenyl.

[0537] In some embodiments, Rlb is a Ci-14 alkyl. In some embodiments, Rlb is a C2-14 alkyl. In some embodiments, Rlb is a C3-14 alkyl. In some embodiments, Rlb is a Ci-8 alkyl. In some embodiments, Rlb is a C1-5 alkyl. In some embodiments, Rlb is a C1-3 alkyl. In some embodiments, Rlb is selected from Ci alkyl, C2 alkyl, C3 alkyl, C4 alkyl, and C5 alkyl. For example, in some embodiments, Rlb is a Ci alkyl. For example, in some embodiments, Rlb is a C2 alkyl. For example, in some embodiments, Rlb is a C3 alkyl. For example, in some embodiments, Rlb is a C4 alkyl. For example, in some embodiments, Rlb is a C5 alkyl.

[0538] In some embodiments, R1 is different from -(CHR5R6)mM-CR2R3R7.

[0539] In some embodiments, -CHR1aRlb- is different from -(CHR5R6)mM-CR2R3R7.

[0540] In some embodiments, R7 is H. In some embodiments, R7 is selected from C1-3 alkyl. For example, in some embodiments, R7 is Ci alkyl. For example, in some embodiments, R7 is C2 alkyl. For example, in some embodiments, R7 is C3 alkyl. In some embodiments, R7 is selected from C4 alkyl, C4 alkenyl, C5 alkyl, C5 alkenyl, Ce alkyl, Ce alkenyl, C7 alkyl, C7 alkenyl, C9 alkyl, C9 alkenyl, C11 alkyl, C11 alkenyl, C17 alkyl, C17 alkenyl, C1e alkyl and C1e alkenyl.

[0541] In some embodiments, Rb is Ci-i4 alkyl. In some embodiments, Rb is C2-14 alkyl. In some embodiments, Rb is C3-14 alkyl. In some embodiments, Rb is Ci-8 alkyl. In some embodiments, Rb is C1-5 alkyl. In some embodiments, Rb is C1-3 alkyl. In some embodiments, Rb is selected from Ci alkyl, C2 alkyl, C3 alkyl, C4 alkyl and C5 alkyl. For example, in some embodiments, Rb is Ci alkyl. For example, in some embodiments, Rb is C2 alkyl. For example, in some embodiments, Rb is C3 alkyl. For example, in some embodiments, Rb is C4 alkyl.

[0542] In some embodiments, the compound of Formula (I) has the formula (IIa): [ka] wherein R4 is as described herein. or an N-oxide or salt or isomer thereof.

[0543] In other embodiments, the compound of Formula (I) has the formula (IIb): [ka] wherein R4 is as described herein. or an N-oxide or salt or isomer thereof.

[0544] In other embodiments, the compound of formula (I) has the formula (lie) or the formula (He): [ka] wherein R4 is as described herein. or an N-oxide or salt or isomer thereof.

[0545] In other embodiments, the compound of Formula (I) has the formula (IIf): [ka] [During the ceremony, M is -C(O)O- or -OC(O)-, M" is C1-6 alkyl or C2-6 alkenyl, R2 and R3 are independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl, and n is selected from 2, 3, and 4. or an N-oxide or salt or isomer thereof.

[0546] In a further embodiment, the compound of formula (I) has the formula (lid): [ka] [During the ceremony, n is 2, 3, or 4; m, R', R'', and R2-R6 are as described herein. or an N-oxide, salt, or isomer thereof. For example, each of R2 and R3 can be independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.

[0547] In a further embodiment, the compound of formula (I) has the formula (IIg): [ka] [During the ceremony, l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; Mi is a bond or M'; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M''-C(O)O-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl. or an N-oxide or salt or isomer thereof. For example, M" is Ci-6 alkyl (e.g., C1-4 alkyl) or C2-6 alkenyl (e.g., C2-4 alkenyl). For example, R2 and R3 are independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl. or an N-oxide or salt or isomer thereof.

[0548] In other embodiments, some of the compounds of formula (VI) have the formula (Vila): [ka] or an N-oxide thereof or a salt or isomer thereof.

[0549] In other embodiments, some of the compounds of formula (VI) are represented by formula (Villa): [ka] or an N-oxide thereof or a salt or isomer thereof.

[0550] In other embodiments, some of the compounds of formula (VI) have the formula (VIIIb): [ka] or an N-oxide thereof or a salt or isomer thereof.

[0551] In other embodiments, some of the compounds of formula (VI) have the formula (Vllb-1): [ka] or an N-oxide thereof or a salt or isomer thereof.

[0552] In other embodiments, a compound of formula (VIIb-2): [ka] or an N-oxide thereof or a salt or isomer thereof.

[0553] In other embodiments, some of the compounds of formula (VI) are represented by formula ...

Claims

1. 1. A method of delivering a nucleic acid to a primate in need thereof, comprising administering to the primate lipid nanoparticles (LNPs), wherein the LNPs: i) a nucleic acid or a pharmaceutically acceptable salt thereof encapsulated in an LNP; ii) cationic lipids; iii) neutral lipids; iv) steroids; and v) Polymer-conjugated lipids wherein the plurality of LNPs have an average particle size in the range of 40 nm to 70 nm.

2. The method of claim 1, wherein the average particle size is in the range of 50 nm to 70 nm.

3. The method of claim 1, wherein the average particle size is in the range of 55 nm to 65 nm.

4. The method of claim 1, wherein the average particle size is in the range of 50 nm to 60 nm.

5. The method of claim 1, wherein the average particle size is in the range of 60 nm to 70 nm.

6. 2. The method of claim 1, wherein the average particle size is about 47 nm, about 48 nm, about 49 nm, about 50 nm, about 51 nm, about 52 nm, about 53 nm, about 54 nm, about 55 nm, about 56 nm, about 57 nm, about 58 nm, about 59 nm, about 60 nm, about 61 nm, about 62 nm, about 63 nm, about 64 nm or about 65 nm.

7. 1. A method of delivering a nucleic acid to a primate in need thereof, comprising administering to the primate lipid nanoparticles (LNPs), wherein the LNPs: i) a nucleic acid or a pharmaceutically acceptable salt thereof encapsulated in an LNP; ii) cationic lipids; iii) neutral lipids; iv) steroids; and v) 2.0 to 3.5 mole percent of polymer-conjugated lipid based on the total moles in the lipid of the LNP. The method includes:

8. 8. The method of claim 7, wherein the LNPs comprise 2.2 to 3.3 mole percent polymer-conjugated lipid.

9. 8. The method of claim 7, wherein the LNPs comprise 2.3 to 2.8 mole percent polymer-conjugated lipid.

10. 8. The method of claim 7, wherein the LNPs comprise 2.1 to 2.5 mole percent polymer-conjugated lipid.

11. 8. The method of claim 7, wherein the LNPs comprise 2.5 to 2.9 mole percent polymer-conjugated lipid.

12. 8. The method of claim 7, wherein the LNP comprises about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, or about 2.8 mole percent of the polymer-conjugated lipid.

13. The polymer-conjugated lipid has the following structure: 【Chemistry 1】 wherein P is a polymer; L is a trivalent linker 1 to 15 atoms in length; R' and R'' are each independently a saturated alkyl having 8 to 14 carbon atoms. The method according to any one of claims 1 to 12, wherein

14. 14. The method of claim 13, wherein P comprises a polyethylene glycol polymer.

15. 15. The method of claim 14, wherein the polyethylene glycol polymer is a hydroxyl or alkoxyl terminated polyethylene glycol polymer.

16. 16. The method of any one of claims 13 to 15, wherein L comprises an amide, ester and / or carbamate functional group.

17. The polymer-conjugated lipid has the following structure: 【Chemistry 2】 wherein n is an integer ranging from 30 to 60; R' and R'' are each independently a saturated alkyl having 8 to 14 carbon atoms; and R''' is H or C １ -C ６ alkyl] The method according to any one of claims 13 to 16, wherein the method comprises one of the following:

18. The polymer-conjugated lipid has the following structure: 【Transformation 3】 wherein n is an integer ranging from 40 to 50, and each R is a saturated alkyl having 8 to 14 carbon atoms, or 8 to 13 carbon atoms, or 8 carbon atoms, or 9 carbon atoms, or 10 carbon atoms, or 11 carbon atoms, or 12 carbon atoms, or 13 carbon atoms.

18. The method of claim 17, comprising:

19. The polymer-conjugated lipid has the following structure: 【Chemistry 4】 [During the ceremony, R ３ Ha-OR Ｏ and R Ｏ is hydrogen or alkyl; r is an integer ranging from 30 to 60, inclusive; R ５ is C １０－２０ alkyl] The method according to any one of claims 1 to 12, wherein (Claim 18) R ３ is OH or OCH ３ and R ５ is C １８ , C １９ or C ２０ and r is 【Transformation 5】 is selected to have an average molecular weight in the range of 1,800 Da to 2,200 Da The method of claim 17,

20. 1. A method of delivering a nucleic acid to a primate in need thereof, comprising administering to the primate lipid nanoparticles (LNPs), wherein the LNPs: i) a nucleic acid or a pharmaceutically acceptable salt thereof encapsulated in an LNP; ii) cationic lipids; iii) neutral lipids; iv) steroids; and v) a polymer-conjugated lipid having the following structure: 【Transformation 6】 [During the ceremony, P is a polymer; L is a trivalent linker 1 to 15 atoms in length; R' and R'' are each independently saturated alkyl having from 8 to 14 carbon atoms, provided that the total number of carbon atoms overall for both R' and R'' is 27 or less. The method includes:

21. 21. The method of claim 20, wherein P comprises a polyethylene glycol polymer.

22. 22. The method of claim 21, wherein the polyethylene glycol polymer is a hydroxyl or alkoxyl terminated polyethylene glycol polymer.

23. 23. The method of any one of claims 20 to 22, wherein L comprises an amide, ester and / or carbamate functional group.

24. The polymer-conjugated lipid has the following structure: 【Transformation 7】 wherein R''' is H or C １ -C ６ alkyl, and n is an integer ranging from 30 to 60. The method according to any one of claims 20 to 23, wherein the method comprises one of the following:

25. The polymer-conjugated lipid has the following structure: 【Transformation 8】 (wherein n is an integer ranging from 40 to 50) 25. The method of claim 24, wherein

26. The method of any one of claims 20 to 25, wherein the total number of carbon atoms in R' and R'' ranges from 16 to 26, 16 to 24, 17 to 24, or 18 to 24.

27. a) R' and R'' are each saturated alkyl having 8 carbon atoms; b) R' and R'' are each saturated alkyl having 9 carbon atoms; c) R' and R'' are each saturated alkyl having 10 carbon atoms; d) R' and R'' are each saturated alkyl having 11 carbon atoms; e) R' and R'' are each saturated alkyl having 12 carbon atoms; or f) R' and R'' are each saturated alkyl having 13 carbon atoms; The method according to any one of claims 20 to 25.

28. 28. The method of any one of claims 1-6 and 13-27, wherein the LNP comprises 2.0 to 3.0 mole percent polymer-conjugated lipid based on the total moles in the lipid of the LNP.

29. 29. The method of claim 28, wherein the LNPs comprise 2.2 to 3.3 mole percent polymer-conjugated lipid.

30. 29. The method of claim 28, wherein the LNPs comprise 2.3 to 2.8 mole percent polymer-conjugated lipid.

31. 29. The method of claim 28, wherein the LNPs comprise 2.1 to 2.5 mole percent polymer-conjugated lipid.

32. 29. The method of claim 28, wherein the LNPs comprise 2.5 to 2.9 mole percent polymer-conjugated lipid.

33. 29. The method of claim 28, wherein the LNP comprises about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, or about 2.8 mole percent of the polymer-conjugated lipid.

34. 28. The method of any one of claims 7-12 and 20-27, wherein the plurality of LNPs have an average particle size in the range of 40 nm to 70 nm.

35. 35. The method of claim 34, wherein the average particle size is in the range of 50 nm to 70 nm.

36. 35. The method of claim 34, wherein the average particle size is in the range of 55 nm to 65 nm.

37. 35. The method of claim 34, wherein the average particle size is in the range of 50 nm to 60 nm.

38. 35. The method of claim 34, wherein the average particle size is in the range of 60 nm to 70 nm.

39. 35. The method of claim 34, wherein the average particle size is about 47 nm, about 48 nm, about 49 nm, about 50 nm, about 51 nm, about 52 nm, about 53 nm, about 54 nm, about 55 nm, about 56 nm, about 57 nm, about 58 nm, about 59 nm, about 60 nm, about 61 nm, about 62 nm, about 63 nm, about 64 nm or about 65 nm.

40. The cationic lipid has the formula (I): 【Chemistry 9】 [During the ceremony, L １ or L ２ One of the groups is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) ｘ -, -S-S-, -C(=O)S-, SC(=O)-, -NR ａ C(=O)-, -C(=O)NR ａ -, NR ａ C(=O)NR ａ -, -OC(=O)NR ａ -or-NR ａ C(=O)O-, and L １ or L ２ The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) ｘ -, -S-S-, -C(=O)S-, SC(=O)-, -NR ａ C(=O)-, -C(=O)NR ａ -, NR ａ C(=O)NR ａ -, -OC(=O)NR ａ -or-NR ａ C(═O)O— or a direct bond; R ａ is H or C １ -C １２ is alkyl; R １ａ and R １ｂ is, in each occurrence, independently: (a) H or C １ -C １２ alkyl, or (b) R １ａ is H or C １ -C １２ alkyl, and R １ｂ together with the carbon atom to which it is attached, the adjacent R １ｂ and together with the carbon atom to which it is attached form a carbon-carbon double bond; R ２ａ and R ２ｂ is, in each occurrence, independently: (a) H or C １ -C １２ alkyl, or (b) R ２ａ is H or C １ -C １２ alkyl, and R ２ｂ together with the carbon atom to which it is attached, the adjacent R ２ｂ and together with the carbon atom to which it is attached form a carbon-carbon double bond; R ３ａ and R ３ｂ is, in each occurrence, independently: (a) H or C １ -C １２ alkyl, or (b) R ３ａ is H or C １ -C １２ alkyl, and R ３ｂ together with the carbon atom to which it is attached, the adjacent R ３ｂ and together with the carbon atom to which it is attached form a carbon-carbon double bond; R ４ａ and R ４ｂ is, in each occurrence, independently: (a) H or C １ -C １２ alkyl, or (b) R ４ａ is H or C １ -C １２ alkyl, and R ４ｂ together with the carbon atom to which it is attached, the adjacent R ４ｂ and together with the carbon atom to which it is attached form a carbon-carbon double bond; R ５ and R ６ are each independently methyl or cycloalkyl; R ７ is, in each occurrence, independently H or C １ -C １２ is alkyl; R ８ and R ９ are each independently an unsubstituted C １ -C １２ alkyl; or R ８ and R ９ together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocyclic ring containing one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; e is 1 or 2; x is 0, 1 or 2.

40. The method of any one of claims 1 to 39, wherein the compound has the structure:

41. The cationic lipid has the formula (II): 【Chemistry 10】 [During the ceremony, L １ or L ２ One of the groups is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) ｘ -, -S-S-, -C(=O)S-, SC(=O)-, -NR ａ C(=O)-, -C(=O)NR ａ -, NR ａ C(=O)NR ａ -, -OC(=O)NR ａ -or-NR ａ C(=O)O-, and L １ or L ２ The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) ｘ -, -S-S-, -C(=O)S-, -SC(=O)-, -NR ａ C(=O)-, -C(=O)NR ａ -, NR ａ C(=O)NR ａ -, -OC(=O)NR ａ -or-NR ａ C(═O)O— or a direct bond; G １ is C １ -C ２ Alkylene, —(C═O)—, —O(C═O)—, —SC(═O)—, —NR ａ C(═O)— or a direct bond; G ２ -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NR ａ - or a direct bond; G ３ is C １ -C ６ alkylene; R ａ is H or C １ -C １２ is alkyl; R １ａ and R １ｂ is, in each occurrence, independently: (a) H or C １ -C １２ alkyl; or (b) R １ａ is H or C １ -C １２ alkyl, and R １ｂ together with the carbon atom to which it is attached, the adjacent R １ｂ and together with the carbon atom to which it is attached form a carbon-carbon double bond; R ２ａ and R ２ｂ is, in each occurrence, independently: (a) H or C １ -C １２ alkyl; or (b) R ２ａ is H or C １ -C １２ alkyl, and R ２ｂ together with the carbon atom to which it is attached, the adjacent R ２ｂ and together with the carbon atom to which it is attached form a carbon-carbon double bond; R ３ａ and R ３ｂ is, in each occurrence, independently (a): H or C １ -C １２ alkyl; or (b) R ３ａ is H or C １ -C １２ alkyl, and R ３ｂ together with the carbon atom to which it is attached, the adjacent R ３ｂ and together with the carbon atom to which it is attached form a carbon-carbon double bond; R ４ａ and R ４ｂ is, in each occurrence, independently: (a) H or C １ -C １２ alkyl; or (b) R ４ａ is H or C １ -C １２ alkyl, and R ４ｂ together with the carbon atom to which it is attached, the adjacent R ４ｂ and together with the carbon atom to which it is attached form a carbon-carbon double bond; R ５ and R ６ are each independently H or methyl; R ７ is C ４ -C ２０ is alkyl; R ８ and R ９ are each independently １ -C １２ alkyl; or R ８ and R ９ together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; x is 0, 1 or 2.

40. The method of any one of claims 1 to 39, wherein the compound has the structure:

42. The cationic lipid has the formula III: 【Chemistry 11】 [During the ceremony, L １ or L ２ One of the groups is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) ｘ -, -S-S-, -C(=O)S-, SC(=O)-, -NR ａ C(=O)-, -C(=O)NR ａ -, NR ａ C(=O)NR ａ -, -OC(=O)NR ａ -or-NR ａ C(=O)O-, and L １ or L ２ The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) ｘ -, -S-S-, -C(=O)S-, SC(=O)-, -NR ａ C(=O)-, -C(=O)NR ａ -, -NR ａ C(=O)NR ａ -, -OC(=O)NR ａ -or-NR ａ C(═O)O— or a direct bond; G １ and G ２ are each independently an unsubstituted C １ -C １２ Alkylene or C １ -C １２ alkenylene; G ３ is C １ -C ２４ Alkylene, C １ -C ２４ Alkenylene, C ３ -C ８ Cycloalkylene, C ３ -C ８ is cycloalkenylene; R ａ is H or C １ -C １２ is alkyl; R １ and R ２ are each independently ６ -C ２４ Alkyl or C ６ -C ２４ alkenyl; R ３ is H, OR ５ , CN, -C(=O)OR ４ , -OC(=O)R ４ or -NR ５ C(=O)R ４ and R ４ is C １ -C １２ is alkyl; R ５ is H or C １ -C ６ is alkyl; x is 0, 1 or 2.

40. The method of any one of claims 1 to 39, wherein the compound has the structure:

43. The cationic lipid has the following formula (IV): 【Chemistry 12】 [During the ceremony, G １ or G ２ is, in each case, —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) ｙ -, -S-S-, -C(=O)S-, SC(=O)-, -N(R ａ )C(=O)-, -C(=O)N(R ａ )-, -N(R ａ )C(=O)N(R ａ )-, -OC(=O)N(R ａ )- or -N(R ａ )C(=O)O- and G １ or G ２ The other is, in each case, —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) ｙ -, -S-S-, -C(=O)S-, -SC(=O)-, -N(R ａ )C(=O)-, -C(=O)N(R ａ )-, -N(R ａ )C(=O)N(R ａ )-, -OC(=O)N(R ａ )- or -N(R ａ )C(═O)O— or a direct bond; L, at each occurrence, is ∼O(C=O)-, where ∼ represents a covalent bond to X; X is CR ａ and When n is 1, Z is an alkyl, cycloalkyl, or monovalent moiety containing at least one polar functional group; or when n is greater than 1, Z is an alkylene, cycloalkylene, or polyvalent moiety containing at least one polar functional group; R ａ is, in each occurrence, independently: H, C １ -C １２ Alkyl, C １ -C １２ Hydroxyl alkyl, C １ -C １２ Aminoalkyl, C １ -C １２ Alkylaminylalkyl, C １ -C １２ Alkoxyalkyl, C １ -C １２ Alkoxycarbonyl, C １ -C １２ Alkylcarbonyloxy, C １ -C １２ Alkylcarbonyloxyalkyl or C １ -C １２ alkylcarbonyl; R, at each occurrence, is independently: (a) H or C １ -C １２ alkyl; or (b) R, together with the carbon atom to which it is attached, forms a carbon-carbon double bond with an adjacent R and the carbon atom to which it is attached; R １ and R ２ are, in each case, respectively, of the following structure: 【Chemistry 13】 a １ and a ２ is, in each occurrence, independently an integer from 3 to 12; b １ and b ２ is, in each occurrence, independently 0 or 1; c １ and c ２ is, in each occurrence, independently an integer from 5 to 10; d １ and d ２ is, in each occurrence, independently an integer from 5 to 10; y, in each occurrence, is independently an integer from 0 to 2; n is an integer ranging from 1 to 6; wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl, and alkylcarbonyl may optionally be substituted with one or more substituents.

40. The method of any one of claims 1 to 39, wherein the compound has the structure:

44. The cationic lipid has the following formula (V): 【Chemistry 14】 [During the ceremony, G １ or G ２ is, in each case, —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) ｙ -, -S-S-, -C(=O)S-, SC(=O)-, -N(R ａ )C(=O)-, -C(=O)N(R ａ )-, -N(R ａ )C(=O)N(R ａ )-, -OC(=O)N(R ａ )- or -N(R ａ )C(=O)O-, and G １ or G ２ The other is, in each case, —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) ｙ -, -S-S-, -C(=O)S-, -SC(=O)-, -N(R ａ )C(=O)-, -C(=O)N(R ａ )-, -N(R ａ )C(=O)N(R ａ )-, -OC(=O)N(R ａ )- or -N(R ａ )C(═O)O— or a direct bond; L, at each occurrence, is ∼O(C=O)-, where ∼ represents a covalent bond to X; X is CR ａ and When n is 1, Z is an alkyl, cycloalkyl, or monovalent moiety containing at least one polar functional group; or when n is greater than 1, Z is an alkylene, cycloalkylene, or polyvalent moiety containing at least one polar functional group; R ａ is, in each occurrence, independently: H, C １ -C １２ Alkyl, C １ -C １２ Hydroxyl alkyl, C １ -C １２ Aminoalkyl, C １ -C １２ Alkylaminylalkyl, C １ -C １２ Alkoxyalkyl, C １ -C １２ Alkoxycarbonyl, C １ -C １２ Alkylcarbonyloxy, C １ -C １２ Alkylcarbonyloxyalkyl or C １ -C １２ alkylcarbonyl; R, at each occurrence, is independently: (a) H or C １ -C １２ alkyl; or (b) R, together with the carbon atom to which it is attached, forms a carbon-carbon double bond with an adjacent R and the carbon atom to which it is attached; R １ and R ２ are, in each case, respectively, of the following structure: 【Chemistry 15】 [During the ceremony, R', at each occurrence, is independently H or C １ -C １２ is alkyl; a １ and a ２ is, in each occurrence, independently an integer from 3 to 12; b １ and b ２ is, in each occurrence, independently 0 or 1; c １ and c ２ is, in each occurrence, independently an integer from 2 to 12; d １ and d ２ is, in each occurrence, independently an integer from 2 to 12; y, in each occurrence, is independently an integer from 0 to 2; n is an integer ranging from 1 to 6; Here, a １ , a ２ , c １ , c ２ , d １ and d ２ is a １ +c １ +d １ is an integer ranging from 18 to 30, ２ +c ２ +d ２ is selected so that the sum of is an integer in the range of 18 to 30, wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl, and alkylcarbonyl may optionally be substituted with one or more substituents.

40. The method of any one of claims 1 to 39, wherein the compound has the formula: or a pharmaceutically acceptable salt or stereoisomer thereof.

45. The cationic lipid has the following formula (VI): 【Chemistry 16】 [During the ceremony, L １ and L ２ are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, or -S(O) ｘ -, -S-S-, -C(=O)S-, -SC(=O)-, -NR ａ C(=O)-, -C(=O)NR ａ -, -NR ａ C(=O)NR ａ -, -OC(=O)NR ａ -, -NR ａ C(═O)O— or a direct bond; G １ is C １ -C ２ Alkylene, —(C═O)—, —O(C═O)—, —SC(═O)—, —NR ａ C(═O)— or a direct bond; G ２ -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NR ａ - or a direct bond; G ３ is C １ -C ６ alkylene; R ａ is H or C １ -C １２ is alkyl; R １ａ and R １ｂ is, in each occurrence, independently: (a) H or C １ -C １２ alkyl; or (b) R １ａ is H or C １ -C １２ alkyl, and R １ｂ together with the carbon atom to which it is attached, the adjacent R １ｂ and together with the carbon atom to which it is attached form a carbon-carbon double bond; R ２ａ and R ２ｂ is, in each occurrence, independently: (a) H or C １ -C １２ alkyl; or (b) R ２ａ is H or C １ -C １２ alkyl, and R ２ｂ together with the carbon atom to which it is attached, the adjacent R ２ｂ and together with the carbon atom to which it is attached form a carbon-carbon double bond; R ３ａ and R ３ｂ is, in each occurrence, independently (a): H or C １ -C １２ alkyl; or (b) R ３ａ is H or C １ -C １２ alkyl, and R ３ｂ together with the carbon atom to which it is attached, the adjacent R ３ｂ and together with the carbon atom to which it is attached form a carbon-carbon double bond; R ４ａ and R ４ｂ is, in each occurrence, independently: (a) H or C １ -C １２ alkyl; or (b) R ４ａ is H or C １ -C １２ alkyl, and R ４ｂ together with the carbon atom to which it is attached, the adjacent R ４ｂ and together with the carbon atom to which it is attached form a carbon-carbon double bond; R ５ and R ６ are each independently H or methyl; R ７ is H or C １ -C ２０ is alkyl; R ８ is OH, -N(R ９ )(C=O)R １０ , -(C=O)NR ９ R １０ , -NR ９ R １０ , -(C=O)OR １１ or -O(C=O)R １１ where R ８ Ga-NR ９ R １０ When G ３ is C ４ -C ６ alkylene; R ９ and R １０ are each independently H or C １ -C １２ is alkyl; R １１ is aralkyl; a, b, c and d are each independently an integer from 1 to 24; x is 0, 1 or 2; Each alkyl, alkylene and aralkyl may be optionally substituted.

40. The method of any one of claims 1 to 39, wherein the compound has the formula: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

46. The cationic lipid has the following formula (VII): 【Chemistry 17】 [During the ceremony, X and X′ are each independently N or CR; Y and Y′ are each independently absent, —O(C═O)—, —(C═O)O—, or NR, with the proviso that: a) when X is N, Y is absent; b) when X' is N, Y' is absent; c) when X is CR, Y is —O(C═O)—, —(C═O)O—, or NR; d) when X' is CR, Y' is -O(C=O)-, -(C=O)O- or NR; L １ and L １ Each ' is independently -O(C=O)R １ , -(C=O)OR １ , -C(=O)R １ , -OR １ , -S(O) ｚ R １ , -S-SR １ , -C(=O)SR １ , -SC(=O)R １ , -NR ａ C(=O)R １ , —C(═O)NR ｂ R ｃ , -NR ａ C(=O)NR ｂ R ｃ , —OC(═O)NR ｂ R ｃ or -NR ａ C(=O)OR １ and L ２ and L ２ Each ' is independently -O(C=O)R ２ , -(C=O)OR ２ , -C(=O)R ２ , -OR ２ , -S(O) ｚ R ２ , -S-SR ２ , -C(=O)SR ２ , -SC(=O)R ２ , -NR ｄ C(=O)R ２ , —C(═O)NR ｅ R ｆ , -NR ｄ C(=O)NR ｅ R ｆ , —OC(═O)NR ｅ R ｆ ;-NR ｄ C(=O)OR ２ or R ２ is a direct bond to G １ , G １ ', G ２ and G ２ Each ' is independently C ２ -C １２ Alkylene or C ２ -C １２ alkenylene; G ３ is C ２ -C ２４ Heteroalkylene or C ２ -C ２４ heteroalkenylene; R ａ , R ｂ , R ｄ and R ｅ is, in each occurrence, independently: H, C １ -C １２ Alkyl or C ２ -C １２ alkenyl; R ｃ and R ｆ is, in each occurrence independently, C １ -C １２ Alkyl or C ２ -C １２ alkenyl; R, in each occurrence, is independently H or C １ -C １２ is alkyl; R １ and R ２ is, in each occurrence independently, a branch C ６ -C ２４ Alkyl or branched C ６ -C ２４ alkenyl; z is 0, 1 or 2; wherein, unless otherwise specified, each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene, and heteroalkenylene is independently substituted or unsubstituted.

40. The method of any one of claims 1 to 39, wherein the compound has the formula: or a pharmaceutically acceptable salt or stereoisomer thereof.

47. The cationic lipid has the following formula (VIII): [Chemistry 18] [During the ceremony, X is N and Y is absent; or X is CR and Y is NR; L １ is -O(C=O)R １ , -(C=O)OR １ , -C(=O)R １ , -OR １ , -S(O) ｘ R １ , -S-SR １ , -C(=O)SR １ , -SC(=O)R １ , -NR ａ C(=O)R １ , —C(═O)NR ｂ R ｃ , -NR ａ C(=O)NR ｂ R ｃ , —OC(═O)NR ｂ R ｃ or -NR ａ C(=O)OR １ and L ２ is -O(C=O)R ２ , -(C=O)OR ２ , -C(=O)R ２ , -OR ２ , -S(O) ｘ R ２ , -S-SR ２ , -C(=O)SR ２ , -SC(=O)R ２ , -NR ｄ C(=O)R ２ , —C(═O)NR ｅ R ｆ , -NR ｄ C(=O)NR ｅ R ｆ , —OC(═O)NR ｅ R ｆ ;-NR ｄ C(=O)OR ２ or R ２ is a direct bond to L ３ is -O(C=O)R ３ or -(C=O)OR ３ and G １ and G ２ are each independently ２ -C １２ Alkylene or C ２ -C １２ alkenylene; When X is CR and Y is NR, G ３ is C １ -C ２４ Alkylene, C ２ -C ２４ Alkenylene, C １ -C ２４ Heteroalkylene or C ２ -C ２４ heteroalkenylene; when X is N and Y is absent, G ３ is C １ -C ２４ Heteroalkylene or C ２ -C ２４ heteroalkenylene; R ａ , R ｂ , R ｄ and R ｅ are each independently H or C １ -C １２ Alkyl or C １ -C １２ alkenyl; R ｃ and R ｆ are each independently １ -C １２ Alkyl or C ２ -C １２ alkenyl; Each R is independently H or C １ -C １２ is alkyl; R １ , R ２ and R ３ are each independently １ -C ２４ Alkyl or C ２ -C ２４ alkenyl; x is 0, 1 or 2; wherein, unless otherwise specified, each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene, and heteroalkenylene is independently substituted or unsubstituted.

40. The method of any one of claims 1 to 39, wherein the compound has the formula: or a pharmaceutically acceptable salt or stereoisomer thereof.

48. The cationic lipid has the following formula (IX): 【Chemistry 19】 [During the ceremony, L １ is -O(C=O)R １ , -(C=O)OR １ , -C(=O)R １ , -OR １ , -S(O) ｘ R １ , -S-SR １ , -C(=O)SR １ , -SC(=O)R １ , -NR ａ C(=O)R １ , —C(═O)NR ｂ R ｃ , -NR ａ C(=O)NR ｂ R ｃ , —OC(═O)NR ｂ R ｃ or -NR ａ C(=O)OR １ and L ２ is -O(C=O)R ２ , -(C=O)OR ２ , -C(=O)R ２ , -OR ２ , -S(O) ｘ R ２ , -S-SR ２ , -C(=O)SR ２ , -SC(=O)R ２ , -NR ｄ C(=O)R ２ , —C(═O)NR ｅ R ｆ , -NR ｄ C(=O)NR ｅ R ｆ , —OC(═O)NR ｅ R ｆ ;-NR ｄ C(=O)OR ２ or R ２ is a direct bond to G １ and G ２ are each independently ２ -C １２ Alkylene or C ２ -C １２ alkenylene; G ３ is C １ -C ２４ Alkylene, C ２ -C ２４ Alkenylene, C ３ -C ８ Cycloalkylene or C ３ -C ８ is cycloalkenylene; R ａ , R ｂ , R ｄ and R ｅ are each independently H or C １ -C １２ Alkyl or C １ -C １２ alkenyl; R ｃ and R ｆ are each independently １ -C １２ Alkyl or C ２ -C １２ alkenyl; R １ and R ２ each independently represents a branch C ６ -C ２４ Alkyl or branched C ６ -C ２４ alkenyl; R ３ HA-N(R ４ )R ５ and R ４ is C １ -C １２ is alkyl; R ５ is a substitution C １ -C １２ is alkyl; x is 0, 1 or 2; wherein, unless otherwise specified, each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl, and aralkyl is independently substituted or unsubstituted.

40. The method of any one of claims 1 to 39, wherein the compound has the formula: or a pharmaceutically acceptable salt or stereoisomer thereof.

49. The cationic lipid has the following formula (X): 【Chemistry 20】 [During the ceremony, G １ -OH, -NR ３ R ４ , -(C=O)NR ５ or -NR ３ (C=O)R ５ and G ２ Ha-CH ２ - or -(C=O)-; R, at each occurrence, is independently H or OH; R １ and R ２ each independently represents an optionally substituted branched saturated or unsaturated C １２ -C ３６ is alkyl; R ３ and R ４ are each independently H or an optionally substituted linear or branched saturated or unsaturated C １ -C ６ is alkyl; R ５ is an optionally substituted linear or branched saturated or unsaturated C １ -C ６ is alkyl; n is an integer ranging from 2 to 6.

40. The method of any one of claims 1 to 39, wherein the compound has the formula: or a pharmaceutically acceptable salt or stereoisomer thereof.

50. 18. The method of any one of claims 1 to 17, wherein the cationic lipid is selected from the lipids in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11 or Table 12.

51. 51. The method of any one of claims 1 to 50, wherein the molar ratio of cationic lipid to neutral lipid ranges from about 2:1 to about 8:

1.

52. Neutral lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl 52. The method of any one of claims 1 to 51, wherein the glycerolipid-containing phosphatidylethanolamine is selected from the group consisting of 1-stearoyl-2-oleoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoyl phosphatidylethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (transDOPE).

53. 53. The method of any one of claims 1 to 52, wherein the neutral lipid is DSPC, DPPC, DMPC, DOPC, POPC, DOPE or SM.

54. 54. The method of claim 53, wherein the neutral lipid is DSPC.

55. 55. The method of any one of claims 1 to 54, wherein the steroid is cholesterol.

56. 56. The method of any one of claims 1 to 55, wherein the ratio of cationic lipid to steroid is in the range of 5:1 to 1:

1.

57. 57. The method of any one of claims 1 to 56, wherein the ratio of cationic lipid to polymer-conjugated lipid ranges from about 100:1 to about 20:

1.

58. 58. The method of any one of claims 1 to 57, wherein the nucleic acid is selected from antisense and messenger RNA.

59. 59. The method of claim 58, wherein the nucleic acid comprises an mRNA capable of translating an immunogenic protein.

60. 60. The method of any one of claims 1 to 59, wherein administering comprises intravenous administration.

61. The following structure: 【Chemistry 21】 wherein R′ and R″ are each independently saturated alkyl having from 8 to 12 carbon atoms, provided that the total number of carbon atoms overall for both R′ and R″ is 23 or less; R''' is H or C １ -C ６ is alkyl; n is an integer ranging from 30 to 60. or a salt thereof.

62. 62. The compound of claim 61, wherein n is an integer ranging from 40 to 50.

63. R''' is H or CH ３ 63. The compound of claim 61 or 62,

64. 64. The compound of any one of claims 61-63, wherein the total number of carbon atoms across both R' and R'' is in the range of 16-22, 16-21, 16-20, 18-23, 18-22, 18-21, 19-23, 19-22, 19-21, 20-23, or 20-22.

65. a) R' and R'' are each saturated alkyl having 8 carbon atoms; b) R' and R'' are each saturated alkyl having 9 carbon atoms; c) R' and R'' are each saturated alkyl having 10 carbon atoms; or d) R' and R'' are each saturated alkyl having 11 carbon atoms; 65. The compound according to any one of claims 61 to 64.

66. A lipid nanoparticle comprising a compound according to any one of claims 61 to 65.

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