Fluorinated cationic lipids for lipid nanoparticles

Abstract

The structure: TIFF2024546951000042.tif2132(I) or a pharma- ceutically acceptable salt, tautomer or stereoisomer thereof, wherein R 3 , L 1 , L 2 , G 1 , G 2 and G 3 is as defined herein. Also provided are the use of said compounds as components of lipid nanoparticle formulations for therapeutic agent delivery, compositions comprising said compounds, and methods of use and preparation thereof.

Description

[Technical field]

[0001] The present disclosure generally relates to novel fluorinated cationic lipids that can be used in combination with other lipid components, such as neutral lipids, cholesterol, and polymer-conjugated lipids, to form lipid nanoparticles with oligonucleotides that facilitate intracellular delivery of nucleic acid therapeutics (e.g., oligonucleotides, messenger RNA) both in vitro and in vivo. [Background technology]

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

[0003] Some nucleic acids, such as miRNA inhibitors, can be used to cause the expression of specific cellular products controlled by miRNA, which can be useful for treating diseases related to protein or enzyme deficiency.The therapeutic application of miRNA inhibition is very broad, since constructs can be synthesized to inhibit one or more miRNAs, which in turn controls the expression of mRNA products.The inhibition of endogenous miRNA can increase the expression of its downstream target endogenous protein in cells or organisms and restore proper function, as a means to treat diseases related to specific miRNA or a group of miRNAs.

[0004] Other nucleic acids can downregulate the intracellular levels of specific mRNAs, and thus the synthesis of corresponding proteins, through processes such as RNA interference (RNAi) or complementary binding of antisense RNA. The therapeutic applications of antisense oligonucleotides and RNAi are also extremely broad, since oligonucleotide constructs can be synthesized with any nucleotide sequence directed to target mRNAs. Targets can include mRNAs from normal cells, mRNAs associated with disease states such as cancer, and mRNAs of infectious agents such as viruses. To date, antisense oligonucleotide constructs have demonstrated the ability to specifically downregulate target proteins through the degradation of cognate mRNAs in both in vitro and in vivo models. In addition, antisense oligonucleotide constructs are currently being evaluated in clinical studies.

[0005] However, the use of oligonucleotides in therapeutic settings currently faces two challenges. First, free RNA is susceptible to nuclease digestion in plasma. Second, free RNA has limited ability to access 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 block RNA degradation in plasma and promote cellular uptake of oligonucleotides.

[0006] There remains a need for improved cationic lipids and lipid nanoparticles for oligonucleotide delivery. Preferably, these lipid nanoparticles provide an optimal drug:lipid ratio, protect the nucleic acid from degradation and clearance in serum, are suitable for systemic or local delivery, and provide intracellular delivery of nucleic acid. In addition, these lipid-nucleic acid particles should be well tolerated and provide a sufficient therapeutic index so that treatment of a patient with an effective amount of nucleic acid is not accompanied by unacceptable toxicity and / or risk to said patient. The present invention provides these and related advantages. Summary of the Invention [Means for solving the problem]

[0007] In summary, the present invention provides fluorinated lipid compounds (including their stereoisomers, pharma- ceutically acceptable salts or tautomers) that can be used alone or in combination with other lipid components, such as neutral lipids, charged lipids, steroids (including, for example, all sterols) and / or their analogs, and / or polymer-conjugated lipids, to form lipid nanoparticles for delivery of therapeutic agents. In some cases, lipid nanoparticles are used to deliver nucleic acids, such as antisense and / or messenger RNA. Methods of using such lipid nanoparticles for the treatment of various diseases or conditions, such as those caused by infectious agents and / or protein deficiencies, are also provided.

[0008] In one embodiment, the compound has the following structure (I): [ka] (I) or a pharma- ceutically acceptable salt, tautomer or stereoisomer thereof is provided. [In the formula, R 3 , L 1 , L 2 , G 1 , G 2 and G 3 is as defined herein].

[0009] Also provided is a pharmaceutical composition comprising one or more of the compounds of structure (I) above and a therapeutic agent. In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Such compositions are useful for forming lipid nanoparticles for therapeutic agent delivery.

[0010] In another embodiment, the present invention provides a method of administering a therapeutic agent to a patient in need thereof, comprising preparing a lipid nanoparticle composition comprising a compound of structure (I) and a therapeutic agent, and delivering said composition to the patient.

[0011] These and other aspects of the present invention will become evident upon reference to the following detailed description. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 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] The present invention is based in part on the discovery of novel cationic (amino) lipids that provide advantages when used in lipid nanoparticles for the in vivo delivery of active agents or therapeutic agents, such as nucleic acids, to mammalian cells. In particular, embodiments of the present invention provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein that provide increased activity of the nucleic acid and improved tolerability of the compositions in vivo, resulting in a significantly increased therapeutic index compared to previously described nucleic acid-lipid nanoparticle compositions.

[0014] In certain embodiments, the present invention provides novel cationic lipids that allow for the formulation of improved compositions 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 nanoparticle compositions are useful for upregulating endogenous protein expression by delivering miRNA inhibitors that target a specific miRNA or a group of miRNAs that control a target mRNA or several mRNAs. In other embodiments, these improved lipid nanoparticle compositions are useful for downregulating (e.g., silencing) protein and / or mRNA levels of target genes. In some other embodiments, the lipid nanoparticles are also useful for the delivery of mRNA and plasmids for transgene expression. In still other embodiments, the lipid nanoparticle compositions are useful for inducing pharmacological effects resulting from protein expression, such as increased red blood cell production via delivery of appropriate erythropoietin mRNA, or protection from infection via delivery of appropriate antigen or antibody-encoding mRNA.

[0015] The lipid nanoparticles and compositions of the invention can be used for a variety of purposes, including delivery of encapsulated or associated (e.g., complexed) therapeutic agents, such as, for example, nucleic acids, to cells, both in vitro and in vivo. Accordingly, an embodiment of the invention provides a method of treating or preventing a disease or disorder in a subject in need of treatment by contacting the subject with lipid nanoparticles that encapsulate or associate with a suitable therapeutic agent, the lipid nanoparticles comprising one or more of the novel cationic lipids described herein.

[0016] As described herein, embodiments of the lipid nanoparticles of the present invention are particularly useful for delivery of nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomir / antimir), messenger RNA interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc. Thus, the lipid nanoparticles and compositions of the present invention can be used to induce expression of a desired protein both in vitro and in vivo by contacting a cell with lipid nanoparticles comprising one or more of the novel cationic lipids described herein, where the lipid nanoparticles encapsulate or bind the nucleic acid (e.g., messenger RNA or plasmid encoding the desired protein) to be expressed to produce the desired protein, or inhibit a process that terminates mRNA expression (e.g., miRNA inhibitors). Alternatively, the lipid nanoparticles and compositions of the present invention may be used to reduce the expression of target genes and proteins both in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the novel cationic lipids described herein, where the lipid nanoparticles encapsulate or are associated with a nucleic acid (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)) that reduces the expression of the target gene. The lipid nanoparticles and compositions of the present invention 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 that require co-localization of different nucleic acids (e.g., mRNA encoding an appropriate gene-modifying enzyme and a DNA segment(s) for integration into the host genome).

[0017] Nucleic acids for use with the present invention can be prepared according to any available technique. For mRNA, the primary preparation method is, but is not limited to, enzymatic synthesis (also called in vitro transcription), which currently represents the most efficient method of generating long sequence-specific mRNA. In vitro transcription represents a template-directed process of 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 several 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 Vol. 941, Conn GL (ed.), New York, NY Humana Press, 2012).

[0018] Transcription of RNA occurs in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and ribonucleoside triphosphates (rNTPs) of adenosine, guanosine, uridine and cytidine under conditions that support polymerase activity while minimizing possible degradation of purified mRNA transcripts. In vitro transcription can be performed 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; Kamaka, 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 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530: 117-120). See pages 101-114, all of which are incorporated herein by reference.

[0019] 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 isolation of mRNA transcripts are known in the art. Known procedures include phenol / chloroform extraction or precipitation with any alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Additional non-limiting examples of purification procedures 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 vol. 10, pp. 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 syntheses Methods vol. 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, SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0020] Furthermore, although reverse transcription can produce a large amount of mRNA, the product may contain some abnormal RNA impurities associated with undesired polymerase activity that may need to be removed from full-length mRNA preparations. These include abortive transcription initiation and short RNAs resulting from 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 demonstrated that these contaminants with dsRNA structure can result in undesired immune stimulatory activity through interaction with various innate immune sensors in eukaryotic cells that recognize specific nucleic acid structures and function to induce strong immune responses. This in turn can dramatically reduce mRNA translation due to reduced protein synthesis during the cellular innate immune response. Therefore, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including, but not limited to, scalable HPLC purification (see, 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, vol. 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 vol. 969 (ed. Rabinovich, PH), 2013). HPLC-purified mRNA has been reported to be translated at much greater levels, especially in primary cells and in vivo.

[0021] A considerable variety of modifications have been described in the art that are used to change certain properties of in vitro transcribed mRNA and improve its usefulness. These include, but are not limited to, modifications of the 5' and 3' ends of the mRNA. Endogenous eukaryotic mRNAs usually contain a cap structure on the 5' end of the mature molecule that plays an important role in mediating the binding of mRNA cap-binding protein (CBP), which in turn is involved in enhancing RNA stability in cells and the efficiency of mRNA translation. Thus, the highest level of protein expression is achieved with 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. Additional modifications include methylation of the 5'-most nucleotide and the penultimate 5'-most nucleotide on the 2' hydroxy group.

[0022] Several different cap structures may be used to generate the 5' cap of in vitro transcribed synthetic mRNA. 5' capping of synthetic mRNA can be performed by co-transcription with a chemical cap analog (i.e., capping during in vitro transcription). For 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 as well as a 3'-O-methyl group. However, up to 20% of the transcripts remain uncapped during this co-transcription process, making the synthetic cap analog non-identical to the 5' cap structure of authentic cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules may also be enzymatically capped post-transcriptionally. These may generate more authentic 5' cap structures that more closely mimic endogenous 5' caps structurally or functionally with enhanced binding of cap-binding proteins, extended half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5' decapping. To enhance mRNA stability and translatability, many synthetic 5' cap analogs have been developed and are known in the art (see, 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 Vol. 969 (ed. Rabinovich, PH), 2013).

[0023] At the 3' end, a long chain of adenine nucleotides (polyA tail) is usually added to the mRNA molecule during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved, liberating a 3' hydroxyl group to which polyA polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. Poly(A) tails have 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 14:373-377; Guhaniyogi, J. and Brewer, G., 2001, Regulation of mRNA stability in mammalian cells, Gene 265:11-23; Dreyfus, M. and Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell 111:611-613).

[0024] Poly(A) tailing of in vitro transcribed mRNA can be achieved using a variety of techniques, including but not limited to cloning of a poly(T) tract into a DNA template or post-transcriptional addition using poly(A) polymerase. The first case allows in vitro transcription of mRNAs with poly(A) tails of defined length, depending on the size of the poly(T) tract, but requires additional manipulation of the template. The latter case involves enzymatic addition of poly(A) tails to in vitro transcribed mRNAs using poly(A) polymerase, which catalyzes the incorporation of adenine residues into the 3' end of the RNA, and does not require additional manipulation of the DNA template, but results in mRNAs with poly(A) tails of heterogeneous length. 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), as well as using commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

[0025] In addition to 5' cap and 3' polyadenylation, other modifications of in vitro transcripts have been reported to provide benefits such as those related to translation efficiency and stability. It is known in the art that pathogenic DNA and RNA can be recognized by various sensors in eukaryotic cells and trigger a strong innate immune response. Since most nucleic acids from natural sources contain modified nucleosides, it has been shown that the ability to distinguish between pathogenic DNA and RNA and self-DNA and RNA is based, at least in part, on structure and nucleoside modifications. In contrast, in vitro synthesized RNA lacks these modifications and therefore becomes immunostimulatory, which in turn can inhibit efficient mRNA translation, as outlined above.Introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus reducing this undesired 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, vol. 10, pp. 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, vol. 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 16:1833-1840. Modified nucleosides and nucleotides used in the synthesis of modified RNA can be prepared, monitored and utilized using general methods and procedures known in the art.A wide variety of nucleotide modifications are available that may be incorporated to some degree into in vitro transcribed mRNA, either alone or in combination with other modified nucleosides (see, e.g., US2012 / 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.

[0026] 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 UTRs (preferably 5' and 3' UTRs can be obtained from cellular or viral RNA), either together or independently, has been shown to increase mRNA stability and translation efficiency of in vitro translated mRNAs (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, Vol. 969 (Rabinovich, PH, ed.), 2013).

[0027] In addition to mRNA, other nucleic acid payloads may be used for the present invention. For oligonucleotides, methods of preparation include, but are not limited to, chemical synthesis and enzymatic or chemical cleavage of long precursors, in vitro transcription as described above, etc. Methods of 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, Vol. 288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005; both of which are incorporated herein by reference).

[0028] For plasmid DNA, preparation for use with the present invention typically involves, but is not limited to, in vitro propagation and isolation of the plasmid DNA in liquid cultures containing the plasmid of interest. The presence of a gene encoding resistance to a particular antibiotic (penicillin, kanamycin, etc.) in the plasmid of interest allows bacteria containing the plasmid of interest to be selectively grown in cultures containing the antibiotic. Methods for isolating plasmid DNA are widely used and known in the art (see, for example, Heilig, J., Elbing, KL and Brent, R., (2001) Large-Scale Preparation of Plasmid DNA, Current Protocols in Molecular Biology. Vol. 41:I Issue:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstrom, S., Bjornestedt,R. and Schmidt, SR (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture, Biotechnol. Bioeng., Vol. 99:557-566; and US 6197553B1). 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 PureYield MaxiPrep (Promega) kits, as well as using commercially available reagents.

[0029] Various exemplary embodiments of the cationic lipids of the present invention, lipid nanoparticles and compositions comprising same, and their use to deliver active agents (e.g., therapeutic agents), such as nucleic acids that regulate gene and protein expression, are described in further detail below.

[0030] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0031] Unless the context specifically requires otherwise, throughout this specification and claims, the word "comprises" and variations thereof, such as "comprises" and "comprising," are intended to be interpreted in an open, inclusive sense, i.e., "including, but not limited to."

[0032] Throughout this specification, reference to "one embodiment" or "an embodiment" means 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 phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0033] 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 this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0034] The phrase "induce expression of a desired protein" refers to the ability of a nucleic acid to increase the expression of a desired protein. To test the degree of protein expression, a test sample (e.g., a cell sample in culture expressing the desired protein) or a test mammalian (e.g., a mammalian such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) is contacted with a nucleic acid (e.g., a nucleic acid combined with a lipid of the present invention). The expression of the desired protein in the test sample or test animal is compared to the expression of the desired protein in a control sample (e.g., a cell sample in culture expressing the desired protein) or a control mammalian (e.g., a mammalian such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, that is not contacted with or administered the nucleic acid. If 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 may be assigned a value of 1.0. In one embodiment, induction of expression of a desired protein is achieved when the ratio of expression of the desired protein in a test sample or test mammal to expression of the desired protein in a control sample or control mammal is greater than 1, for example, 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 know suitable assays for determining the expression level of a protein 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 that can generate fluorescence or luminescence under appropriate conditions.

[0035] The phrase "inhibit expression of a target gene" refers to the ability of a nucleic acid to silence, reduce or inhibit expression of a target gene. To test the degree of gene silencing, a test sample (e.g., a cell sample in culture expressing the target gene) or a test mammalian (e.g., a mammalian such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) is contacted with a nucleic acid that silences, reduces or inhibits expression of the target gene. The expression of the target gene in the test sample or test mammalian is compared with the expression of the target gene in a control sample (e.g., a cell sample in culture expressing the target gene) or a control mammalian (e.g., a mammalian such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model) that is not contacted with or administered the nucleic acid. The expression of the target gene in the control sample or control mammalian may be assigned a value of 100%. In certain embodiments, silencing, inhibition, or reduction of a target gene is achieved when the expression level of the target gene in a test sample or test mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the expression level of the target gene in a control sample or control mammal. In other words, the nucleic acid can silence, reduce or inhibit the expression of the target gene in the test sample or test mammal by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the expression level of the target gene in a control sample or control mammal that has not been contacted with or administered the nucleic acid. Suitable assays for determining the expression level of the target gene include, but are not limited to, 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.

[0036] An "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to produce a desired effect, such as an increase or inhibition of expression of a target sequence, compared to the normal expression level 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 an expression product that is not present in the absence of the nucleic acid. If the expression product is present at a level prior to contact with the nucleic acid, increased expression is achieved when the fold increase over the value obtained with a nucleic acid, such as mRNA, is about 1.05, 1.1, 1.2, 1,3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or more times greater than the control. Inhibition of expression of a target gene or sequence is achieved when the value obtained using a nucleic acid such as an antisense oligonucleotide is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% of the control. Suitable assays for measuring expression of a target gene or sequence include, for example, testing protein or RNA levels using techniques known to those skilled in the art, such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of a suitable reporter protein, as well as phenotypic assays known to those skilled in the art.

[0037] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. DNA may be in the form of an antisense molecule, a plasmid DNA, a cDNA, a PCR product, or a vector. RNA may be in the form of a small hairpin RNA (shRNA), a messenger RNA (mRNA), an antisense RNA, a miRNA, a micRNA, a polyvalent RNA, a dicer substrate RNA, or a viral RNA (vRNA), and combinations thereof. Nucleic acids are synthetic, natural, and unnatural, and include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties as the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise limited, the term "nucleic acid" encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses not only the explicitly specified sequence, but also its variants modified in traditional manner (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the 3-position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group."Base" includes purines and pyrimidines, which include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues, and further include synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0038] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises partial or full-length coding sequences necessary for the production of a polypeptide or a precursor of a polypeptide.

[0039] "Gene product," as used herein, refers to the product of a gene, such as an RNA transcript or a polypeptide.

[0040] The term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, generally characterized by being poorly soluble in water but soluble in many organic solvents. Lipids are usually divided into at least three types: (1) "simple lipids", which include fats and oils as well as waxes; (2) "complex lipids", which include phospholipids and glycolipids; and (3) "derived lipids", such as steroids.

[0041] Steroids have the following carbon skeleton: [ka] It is a compound comprising: Non-limiting examples of steroids include cholesterol.

[0042] "Cationic lipid" refers to a lipid that can be positively charged. Exemplary cationic lipids contain one or more positively charged amino groups. Preferred cationic lipids are ionizable so that they can exist in a positively charged or neutral form depending on the pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. This charge state can affect plasma protein absorption, blood clearance, and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well as the ability to form endosomolytic non-bilayer structures that have important implications for intracellular delivery of nucleic acids (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)).

[0043] 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-(monomethoxypolyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like.

[0044] The term "neutral lipid" refers to any of several lipid species 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), ceramides, and steroids, such as sterols and their derivatives. The neutral lipids may be synthetic or naturally occurring.

[0045] The term "charged lipid" refers to any of several lipid species that exist in a positively or negatively charged form, independent of pH, in a useful physiological range, for example, from about pH 3 to about pH 9. Charged lipids can be synthetic or naturally derived. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammoniumpropane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (e.g., DC-Chol).

[0046] The term "lipid nanoparticle" refers to a particle that comprises one or more compounds of structure (I) or other specific cationic lipids and has at least one dimension on the order of nanometers (e.g., 1-1,000 nm). 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 target site of interest (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 compound of structure (I) and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or in the aqueous space enclosed by part or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other deleterious effects induced by the host organism's or cells' machinery, such as a harmful immune response.

[0047] 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 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or 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, the nucleic acid is resistant to degradation by nucleases in aqueous solution when present in the lipid nanoparticle. Lipid nanoparticles containing nucleic acids and methods for their preparation are disclosed, for example, in U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031, and PCT Publication Nos. WO2013 / 016058 and WO2013 / 086373, the complete disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0048] As used herein, "lipid encapsulated" refers to a lipid nanoparticle that provides an active or therapeutic agent, such as a nucleic acid (e.g., mRNA), either fully encapsulated, partially encapsulated, or both. In some embodiments, the nucleic acid (e.g., mRNA) is fully encapsulated within the lipid nanoparticle.

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

[0050] In the context of nucleic acid-lipid nanoparticles, "serum stable" means that nucleotides are not significantly degraded after exposure to serum or nuclease assays that significantly degrade free DNA or RNA.Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0051] "Systemic delivery" as used herein refers to delivery of a therapeutic product that can result in widespread exposure of an active agent within an organism. Some administration techniques can result in systemic delivery of certain agents, while others cannot. Systemic delivery means that a useful, preferably therapeutic, amount of the agent 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] "Local delivery" as used herein refers to the delivery of an active agent directly to a target site in an organism. For example, an agent can be delivered locally by direct injection into a disease site, such as a tumor, other target site, such as an inflammatory site, or into a target organ, such as the liver, heart, pancreas, kidney, etc. Local delivery can also include techniques of local application or local injection, such as intramuscular, subcutaneous or intradermal injection. Local delivery does not exclude systemic pharmacological action.

[0053] "Alkyl" is a saturated alkyl group having, for example, 1 to 24 carbon atoms (C 24 Alkyl), 6 to 24 carbon atoms (C6 to C 24 Alkyl), 4 to 20 carbon atoms (C4 to C 20 Alkyl), 6 to 16 carbon atoms (C6 to C 16 alkyl), 6 to 9 carbon atoms (C6 to C9 alkyl), 1 to 15 carbon atoms (C1 to C 15 Alkyl), 1 to 12 carbon atoms (C1 to C 12"C1-C8 alkyl", 1-8 carbon atoms (C1-C8 alkyl), or 1-6 carbon atoms (C1-C6 alkyl), or any range or specific value within said ranges, and attached to the remainder of the molecule by a single bond, refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless otherwise specified in this specification, alkyl groups are substituted or unsubstituted.

[0054] "Alkenyl" refers to an alkyl group that is unsaturated (i.e., contains at least one carbon-carbon double bond) and has, for example, 2 to 24 carbon atoms (C 24 Alkenyl, 6 to 24 carbon atoms (C6 to C 24 Alkenyl, 4 to 20 carbon atoms (C4-C 20 Alkenyl, 6 to 16 carbon atoms (C6 to C 16 alkenyl), 6 to 9 carbon atoms (C6 to C9 alkenyl), 2 to 15 carbon atoms (C2 to C 15 Alkenyl, 2 to 12 carbon atoms (C2 to C 12 alkenyl), 2 to 8 carbon atoms (C2-C8 alkenyl), or 2 to 6 carbon atoms (C2-C6 alkenyl), or any range or specific value within said ranges, and attached to the remainder of the molecule by a single bond, refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, e.g., ethenyl, n-propenyl, 1-methylethenyl, n-butenyl, n-pentenyl, 1,1-dimethylethenyl, 3-methylhexenyl, 2-methylhexenyl, etc. Unless stated otherwise in this specification, alkenyl groups are substituted or unsubstituted.

[0055] "Fluoroalkyl" refers to an alkyl group in which one or more hydrogen atoms (H) are replaced with a fluorine atom (F). Fluoroalkyl includes straight or branched chain groups consisting of 1) carbon, hydrogen, and fluorine atoms, or 2) carbon and fluorine atoms. Fluoroalkyl includes, for example, alkyl groups consisting of 1 to 24 carbon atoms (C1 to C2). 24 Fluoroalkyl), 6 to 24 carbon atoms (C6 to C 24 Fluoroalkyl), 4 to 20 carbon atoms (C4 to C 20 Fluoroalkyl), 6 to 16 carbon atoms (C6 to C 16 fluoroalkyl), 6 to 9 carbon atoms (C6 to C9 fluoroalkyl), 1 to 15 carbon atoms (C1 to C 15 fluoroalkyl), 1 to 12 carbon atoms (C1 to C 12 perfluoroalkyl), 1 to 8 carbon atoms (C1-C8 fluoroalkyl), or 1 to 6 carbon atoms (C1-C6 fluoroalkyl), or any range or specific value within said ranges, attached to the remainder of the molecule by a single bond, for example, trifluoromethyl (-CF3), perfluoroethyl (-CF2CF3), perfluoro n-propyl (-(CF2)2CF3), perfluoroisopropyl (-CF(CF3)2), perfluoro n-butyl (-(CF2)3CF3), Examples of perfluoroisobutyl (-CF2CF(CF3)2), perfluorotert-butyl (-C(CF3)3), perfluoro n-hexyl (-(CF2)5CF3), perfluoro n-octyl (-(CF2)7CF3), 2,2,2-trifluoroethyl (-CH2CF3), 4,4,4-trifluoro n-butyl (-(CH2)3CF3), 7,7,7-trifluoro n-heptyl (-(CH2)6CF3), or perfluoro n-heptyl (-(CF2)6CF3). For example, C 12 Fluoroalkyl includes 1,1,1,2,2-pentafluoro-3-dodecane (-CH(CFCF)(CH)CH). 17Fluoroalkyl includes 1,1,1,2,2,3,3,4,4,5,5,6,6,12,12,13,13,14,14,15,15,16,16,17,17,17-hexacosafluoro-9-heptadecane (-CH((CH2)2(CF2)5CF3)2). Unless otherwise stated in the specification, fluoroalkyl groups are substituted or unsubstituted.

[0056] "Fluoroalkenyl" refers to an alkenyl group in which one or more hydrogen atoms (H) are replaced with a fluorine atom (F). Fluoroalkenyl includes straight or branched chain groups consisting of 1) carbon, hydrogen, and fluorine atoms, or 2) carbon and fluorine atoms. Fluoroalkenyl includes, for example, 2-24 carbon atoms (C2-C4). 24 Fluoroalkenyl), 6 to 24 carbon atoms (C6 to C 24 Fluoroalkenyl), 4 to 20 carbon atoms (C4 to C 20 Fluoroalkenyl), 6 to 16 carbon atoms (C6 to C 16 fluoroalkenyl), 6 to 9 carbon atoms (C6 to C9 fluoroalkenyl), 2 to 15 carbon atoms (C2 to C 15 Fluoroalkenyl), 2 to 12 carbon atoms (C2 to C 12perfluoroalkyl (-CF(CF3)2), perfluoron-n-propyl (-(CF(CF3)2), perfluoroisopropyl (-CF(CF3)2), perfluoron-n-butyl (-(CF(CF3)3), perfluoroisopropyl (-CF(CF3)2), perfluoroisopropyl (-CF(CF3)3 ... perfluorobutyl (-CF2CF(CF3)2), perfluorotert-butyl (-C(CF3)3), perfluoro n-hexyl (-(CF2)5CF3), perfluoro n-octyl (-(CF2)7CF3), 2,2,2-trifluoroethyl (-CH2CF3), 4,4,4-trifluoro n-butyl (-(CH2)3CF3), 7,7,7-trifluoro n-heptyl (-(CH2)6CF3), or perfluoro n-heptyl (-(CF2)6CF3). For example, C 12 Fluoroalkyl includes 1,1,1,2,2-pentafluoro-3-dodecane (-CH(CFCF)(CH)CH). 17 Fluoroalkyl includes 1,1,1,2,2,3,3,4,4,5,5,6,6,12,12,13,13,14,14,15,15,16,16,17,17,17-hexacosafluoro-9-heptadecane (-CH((CH2)2(CF2)5CF3)2). Unless otherwise stated in the specification, fluoroalkyl groups are substituted or unsubstituted.

[0057] A "perfluoro substituent" or "perfluoro compound" refers to a linear or branched chain substituent or compound in which each C-H bond has been replaced with a C-F bond. Perfluoro substituents or compounds typically contain only carbon-fluorine (CF) and carbon-carbon (CC) bonds, but in some embodiments, perfluoro substituents or compounds contain heteroatoms and / or functional groups, such as OH, COH, halogens, O, and SOH, with the proviso that the perfluoro substituent or compound does not contain a C-H bond and does contain at least one C-F bond. Perfluoro substituents or compounds can be saturated, e.g., 1-24 carbon atoms (C1-C 24 Perfluoroalkyl), 4 to 20 carbon atoms (C4-C 20 Perfluoroalkyl), 6 to 16 carbon atoms (C6-C 16 perfluoroalkyl), 6 to 9 carbon atoms (C6 to C9 perfluoroalkyl), 1 to 15 carbon atoms (C1 to C 15 Perfluoroalkyl), 1 to 12 carbon atoms (C1 to C 12 perfluoroalkyl), 1 to 8 carbon atoms (C1-C8 perfluoroalkyl), or 1 to 6 carbon atoms (C1-C6 perfluoroalkyl), or any range or specific value within said ranges, and is attached to the remainder of the molecule by a single bond, such as, for example, trifluoromethyl (-CF3), perfluoroethyl (-CF2CF3), perfluoro n-propyl (-(CF2)2CF3), perfluoroisopropyl (-CF(CF3)2), perfluoro n-butyl (-(CF2)3CF3), perfluoroisobutyl (-CF2CF(CF3)2), perfluoro tert-butyl (-C(CF3)3), perfluoro n-hexyl (-(CF2)5CF3), perfluoro n-octyl (-(CF2)7CF3), perfluoro n-heptyl (-(CF2)6CF3), and the like.

[0058] "Alkylene" refers to, for example, an alkylene having 1 to 24 carbon atoms (C1 to C 24 Alkylene, 1 to 15 carbon atoms (C1 to C 15 Alkylene, 1 to 12 carbon atoms (C1 to C 12It refers to a straight or branched divalent hydrocarbon chain having carbon atoms of 1 to 8 carbon atoms (C1-C8 alkylene), 1 to 6 carbon atoms (C1-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), 1 to 2 carbon atoms (C1-C2 alkylene), or any range or specific value within said ranges, which is saturated and consists only of carbon and hydrogen atoms and links the remainder of the molecule to a radical group, e.g., methylene, ethylene, propylene, n-butylene, etc. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one or any two carbons within the chain. Unless otherwise specified herein, the alkylene chain is substituted or unsubstituted.

[0059] "Fluoroalkylene" refers to an alkylene as defined above in which at least one C-H bond is replaced with a C-F bond. Fluoroalkylene includes, for example, alkylenes having 1 to 24 carbon atoms (C1 to C 24 Fluoroalkylene), 1 to 15 carbon atoms (C1 to C 15 Fluoroalkylene), 1 to 12 carbon atoms (C1 to C 12 Fluoroalkylenes include fluoroalkylenes having 1 to 8 carbon atoms (C1 to C8 fluoroalkylenes), 1 to 6 carbon atoms (C1 to C6 fluoroalkylenes), 2 to 4 carbon atoms (C2 to C4 fluoroalkylenes), 1 to 2 carbon atoms (C1 to C2 fluoroalkylenes), or any range or specific value of carbon atoms within said ranges, such as fluoromethylene, fluoroethylene, fluoropropylene, n-fluorobutylene, and the like. The fluoroalkylene chain is attached to the rest of the molecule via a single bond and to the radical group via a single bond. The points of attachment of the fluoroalkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless otherwise stated herein, the fluoroalkylene chain is substituted or unsubstituted.

[0060] "Aryl" refers to a carbocyclic group containing hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For purposes of the present invention, an aryl group may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system and may include fused or bridged ring systems. Aryl groups include, but are not limited to, aryl groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless otherwise stated herein, the term "aryl" or the prefix "ar" (e.g., in "aralkyl") is intended to include aryl groups that are optionally substituted.

[0061] An "alkyl acetal" is a compound of the formula -R a CH(OR b )(OR c ), where R a is alkylene as defined above, and R b and R c are each independently an alkyl or alkenyl as defined above. The alkyl acetal group is, for example, an alkyl acetal group having 1 to 24 carbon atoms (C 24 Alkyl acetals, 6 to 24 carbon atoms (C6 to C 24 Alkyl acetals, 4 to 20 carbon atoms (C4 to C 20 Alkyl acetals, 6 to 16 carbon atoms (C6 to C 16 Alkyl acetals, 6 to 24 carbon atoms (C6 to C 24 alkyl acetals), 6 to 9 carbon atoms (C6 to C9 alkyl acetals), 1 to 15 carbon atoms (C1 to C 15 Alkyl acetals, 1 to 12 carbon atoms (C1 to C 12 alkyl acetal), 1 to 8 carbon atoms (C1 to C8 alkyl acetal), or 1 to 6 carbon atoms (C1 to C6 alkyl acetal). Unless stated otherwise in the specification, an alkyl acetal group may be optionally substituted.

[0062] "Fluoroalkyl acetal" refers to an alkyl acetal as defined above, where R a , R b , and / or R c In the formula (I), at least one C-H bond is replaced with a C-F bond. Exemplary fluoroalkyl acetals include, for example, fluoroalkyl acetals having 1 to 24 carbon atoms (C1 to C 24 Fluoroalkyl acetals, 6 to 24 carbon atoms (C6 to C 24 Alkyl acetals, 4 to 20 carbon atoms (C4 to C 20 Fluoroalkyl acetals, 6 to 16 carbon atoms (C6 to C 16 Fluoroalkyl acetals, 6 to 24 carbon atoms (C6 to C 24 fluoroalkyl acetals), 6 to 9 carbon atoms (C6 to C9 fluoroalkyl acetals), 1 to 15 carbon atoms (C1 to C 15 Fluoroalkyl acetals, 1 to 12 carbon atoms (C1 to C 12 fluoroalkyl acetal), 1 to 8 carbon atoms (C1 to C8 alkyl acetal), or 1 to 6 carbon atoms (C1 to C6 fluoroalkyl acetal). Unless stated otherwise in the specification, a fluoroalkyl acetal group may be optionally substituted.

[0063] "Heterocycle" refers to a stable 3- to 18-membered non-aromatic ring radical which consists of 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless stated otherwise in the specification, a heterocyclyl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system which may include fused or bridged ring systems; and, the nitrogen, carbon, or sulfur atoms of the heterocyclyl group can be optionally oxidized; the nitrogen atom can be optionally quaternized; and the heterocyclyl group can be optionally partially or fully saturated. Examples of such heterocyclyl groups include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise in the specification, heterocyclyl groups may be optionally substituted.

[0064] As used herein, the term "substituted" refers to any of the above groups (e.g., alkyl, alkenyl, fluoroalkyl, fluoroalkenyl, perfluoro substituents, perfluoro compounds, alkylene, fluoroalkylene, aryl, alkyl acetal, fluoroalkyl acetal, and / or heterocycle) in which at least one hydrogen atom has been replaced with a non-hydrogen atom, such as, but not limited to: a halogen atom, such as F, Cl, Br, or I; an oxo group (=O); a hydroxy group (-OH); a carboxyl group (-COH); a C1-C2 alkyl group (-C2H); 12 Alkyl group;-(C=O)OR';-O(C=O)R';-C(=O)R';-OR';-S(O) xR';-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) x NR'R', where R' at each occurrence is independently H or C1-C 15 alkyl, and x is 0, 1, or 2. In some embodiments, the substituent is a C1-C 12 In other embodiments, the substituent is an alkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxy group. In other embodiments, the substituent is an alkoxy group (-OR'). In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group (-NR'R').

[0065] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances 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 and alkyl groups that have no substitution.

[0066] "Prodrug" is intended to denote a compound that can be converted under physiological conditions or by solvolysis to a biologically active compound of the invention. Thus, the term "prodrug" refers to a pharma- ceutically acceptable metabolic precursor of a compound of the invention. A prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to an active compound of the invention. Prodrugs are usually rapidly converted in vivo, for example by hydrolysis in blood, to produce the parent compound of the invention. Prodrug compounds often offer advantages of solubility, tissue compatibility, or sustained release in mammalian organisms (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, pp. 21-24 (Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi, T., et al., ACS Symposium Series, Vol. 14, and Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0067] The term "prodrug" is also intended to include any covalently bonded carriers that release the active compound of the invention in vivo when such prodrug is administered to a mammalian subject. Prodrugs of the compounds of the invention can be prepared by modifying functional groups present in the compounds of the invention such that the modifications are cleaved, either by routine manipulation or in vivo, to the parent compounds of the invention. Prodrugs include compounds of the invention in which a hydroxy, amino or mercapto group is bonded to any group that is cleaved to form a free hydroxy, free amino or free mercapto group, respectively, when a prodrug of the compound of the invention 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 the compounds of the invention.

[0068] The invention disclosed herein is also intended to encompass all pharma- ceutically acceptable compounds of the compounds of structure (I) that are isotopically labeled by replacing one or more atoms with an atom having a different atomic mass or mass number. Examples of isotopes that can 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. These radiolabeled compounds may be useful, for example, to determine or measure the effectiveness of compounds by characterizing the site or mechanism of action, or binding affinity to a pharmacologically important site of action. Certain isotopically labeled compounds of structure (I) or (II), for example those incorporating a radioactive isotope, 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 given their ease of incorporation and available means of detection.

[0069] Heavier isotopes, such as deuterium, i.e. 2 Substitutions such as H may be preferred in some circumstances as they may confer certain therapeutic advantages due to greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements.

[0070] Positron-emitting isotopes, e.g. 11 C. 18 F,15 O, and 13 Substitution with, for example, N, may be useful in positron emission topography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of structure (I) may generally be prepared by processes similar to those described in the Preparations and Examples as presented below, by conventional techniques known to those skilled in the art, or by substituting the appropriate isotopically labeled reagent in place of previously utilized non-labeled reagents.

[0071] The invention disclosed herein is also intended to encompass in vivo metabolic products of the disclosed compounds. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily by enzymatic processes. Thus, the invention includes compounds produced by a process comprising administering a compound of the invention to a mammal for a period of time sufficient to produce a metabolic product thereof. Such products are typically identified by administering a detectable dose of a radiolabeled compound of the invention to an animal, such as a rat, mouse, guinea pig, monkey, or human, allowing a sufficient time for metabolism to occur, and isolating the conversion product from urine, blood, or other biological sample.

[0072] "Stable compound" and "stable structure" are intended 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.

[0073] "Mammals" includes humans and both domestic animals, such as laboratory animals and pets (eg, cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domestic animals, such as wildlife.

[0074] A "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine.

[0075] "Pharmaceutically acceptable salt" includes both acid and base addition salts.

[0076] "Pharmaceutically acceptable acid addition salts" refers to those acids which retain the biological effectiveness and properties of the free base, are not otherwise biologically undesirable, and are anhydrous, non-limiting examples of which include inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic 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, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, It refers to salts formed with 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, undecylenic acid, etc.

[0077] "Pharmaceutically acceptable base addition salt" refers to a salt that retains the biological effectiveness and properties of the free acid and is not biologically or otherwise undesirable. 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.

[0078] Crystallization often produces a solvate of the compound of the present invention. As used herein, the term "solvate" refers to an aggregate that contains one or more molecules of the compound of the present invention and one or more molecules of a solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compound of the present invention may exist as a hydrate, including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, etc., as well as the corresponding solvate forms. The compound of the present invention may be a true solvate, while in other cases, the compound of the present invention may simply retain extraneous water or may be a mixture of water and extraneous solvent.

[0079] A "pharmaceutical composition" refers to a formulation of a compound of the invention with a vehicle generally accepted in the art for the delivery of a biologically active compound to a mammal, such as a human. Such vehicles include any pharma- ceutically acceptable carrier, diluent, or excipient therefor.

[0080] "Effective amount" or "therapeutically effective amount" refers to an amount of the compound of the present invention that, when administered to a mammal, preferably a human, is sufficient to effect treatment in the 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 mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art taking into account his or her knowledge and this disclosure.

[0081] "Treating" or "treatment", as used herein, encompasses the treatment of a disease or condition of interest in a mammal, preferably a human, having the disease or condition of interest; (i) preventing the occurrence of a disease or condition in a mammal, particularly where such mammal is predisposed to, but has not yet been diagnosed as having, the condition; (ii) inhibiting a disease or condition, i.e., arresting its onset; (iii) alleviating the disease or condition, i.e., causing the regression of the disease or condition; or (iv) Relieving symptoms caused by a disease or condition, i.e., relieving pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably or may differ in that a particular disease or condition may not have a known causative agent (so the cause has not yet been discovered) and thus is not yet recognized as a disease, but only as an undesirable state or syndrome in which a particular set of symptoms, to a greater or lesser extent, has been identified by clinicians.

[0082] The compounds of the present invention or their pharma- ceutically acceptable salts may contain one or more asymmetric centers and thus may give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be de?ned in terms of absolute stereochemistry as for amino acids as (R)- or (S)-, or (D)- or (L)-. The present invention is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L) isomers may be prepared using chiral synthons or chiral reagents or fractionated using conventional techniques, e.g., chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemates (or racemates of salts or derivatives) using, e.g., chiral high performance liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers, likewise, all tautomeric forms are also intended to be included.

[0083] "Stereoisomers" refer to compounds made up of the same atoms joined by the same bonds, but with different, not interchangeable, three-dimensional structures. The present invention contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0084] "Tautomer" refers to a proton migration from one atom of a molecule to another atom of the same molecule. The invention includes tautomers of any of the above compounds.

[0085] compound In one aspect, the present invention provides novel lipid compounds that can be combined with other lipid components, such as neutral lipids, charged lipids, steroids, and / or polymer-conjugated lipids, to form lipid nanoparticles with oligonucleotides. Without wishing to be bound by theory, it is believed that these lipid nanoparticles shield the therapeutic agent from degradation in serum and provide effective delivery of the therapeutic agent to cells in vitro and in vivo.

[0086] In one embodiment, the compound has the following structure (I): [ka] (I) or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof. [In the formula, L 1 is -O(C=O)R 1a , -(C=O)OR 1a , -C(=O)R 1a , -OR 1a , -S(O) x R 1a , -S-SR 1a , -C(=O)SR 1a , -SC(=O)R 1a , -NR a C(=O)R 1a , -C(=O)NR a R 1a , -NR a C(=O)NR a R 1a , -OC(=O)NR a R 1a , -NR a C(=O)OR 1a or R 1b and; L 2 is -O(C=O)R 2a , -(C=O)OR 2a , -C(=O)R 2a , -OR 2a , -S(O) x R 2a , -S-SR 2a, -C(=O)SR 2a , -SC(=O)R 2a , -NR a C(=O)R 2a , -C(=O)NR a R 2a , -NR a C(=O)NR a R 2a , -OC(=O)NR a R 2a , -NR a C(=O)OR 2a , or R 2b and; G 1 and G 2 are each independently a straight chain or branched chain C1 to C 12 Alkylene or linear or branched C1-C 12 is a fluoroalkylene; G 3 is a straight or branched chain C1-C 12 Alkylene or linear or branched C1-C 12 is a fluoroalkylene; R a are each independently H or C1-C 12 is alkyl; R 1a and R 2a are each independently a branched chain of C6 to C 24 Alkyl, branched chain C6-C 24 Alkenyl, branched C6-C 24 Fluoroalkyl, branched C6-C 24 Fluoroalkenyl, C6-C 24 Alkyl acetal, or C6-C 24 is a fluoroalkyl acetal; R 1b and R 2b are each independently -CH(OR)(OR), where R is each independently a straight or branched C-C 18 Alkyl, straight or branched chain C6-C 18 Alkenyl, straight or branched C6-C 18 Fluoroalkyl, or linear or branched C6-C 18is fluoroalkenyl; R 3 is H, -OR 5 , -CN, -C(=O)OR 4 , -OC(=O)R 4 , -N(R 5 )N 4 , -C(=O)N(R 4 )R 5 , or -NR 5 C(=O)R 4 and R 4 is H, C1~C 12 alkyl or aryl, R 5 is H or C1-C6 alkyl; or R 4 and R 5 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocyclic ring, and Here, G 1 and G 2 At least one of the following is a straight or branched chain C1-C 12 Fluoroalkylene; G 3 is a straight or branched chain C1-C 12 fluoroalkylene; R 1a and R 2a At least one of the following is present: 24 Fluoroalkyl, branched C6-C 24 Fluoroalkenyl, and C6-C 24 fluoroalkyl acetals; and / or R 1b and R 2b At least one of the following is present: linear or branched C6-C 18 Fluoroalkyl and linear or branched C6-C 18 fluoroalkenyl].

[0087] In various embodiments of compound (I), R a are C1 to C 12 It is an alkyl.

[0088] In some embodiments, the compound has the following structure (IA): [ka] (IA) have [In the formula, R 6 is independently H, F, OH, or C1-C for each occurrence. 24 is alkyl; and n is an integer from 1 to 15.

[0089] In some embodiments, the compound has the following structure (IB): [ka] (IB) have [In the formula, y and z are each independently an integer from 1 to 12; and R 7 is independently H or F for each occurrence].

[0090] In any of the foregoing embodiments, L 1 is -O(C=O)R 1a OR - (C=O)OR 1a And L 2 is -O(C=O)R 2a OR - (C=O)OR 2a For example, in some embodiments, L 1 is -O(C=O)R 1a And L 2 is -O(C=O)R 2a In another example, L 1 is -O(C=O)R 1a And L 2 -(C=O)OR 2a In yet another example, L 1 -(C=O)OR 1a And L 2 is -O(C=O)R 2a In yet another example, L 1 -(C=O)OR 1a And L 2 -(C=O)OR 2a It is.

[0091] In some embodiments, the compound has the following structure (IC) or (ID): [ka] has.

[0092] In some embodiments, the compound has the structure (IC): In other embodiments, the compound has the structure (ID).

[0093] In a further embodiment, the compound has the following structure (IE) or (IF): [ka] has.

[0094] In some embodiments, the compound has the structure (IE). In other embodiments, the compound has the structure (IF).

[0095] In some embodiments, n is an integer from 2 to 12. For example, in some embodiments, n is 2, 3, 4, or 5. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5.

[0096] In some embodiments, y and z are each independently an integer from 2 to 10. For example, in some embodiments, y and z are each independently an integer from 4 to 9. In some embodiments, y and z are each independently 5. In some embodiments, y and z are each independently 6. In some embodiments, y and z are each independently 7. In some embodiments, y and z are each independently 8.

[0097] In some embodiments, R 6 is H.

[0098] In some embodiments, R 1and R 2 each independently represents the following structure: [ka] have [In the formula, R 8a and R 8b are independently H, F, C2-C for each occurrence. 16 Alkyl, or C2-C 16 is a fluoroalkyl; a is an integer from 1 to 16; and R 8a , R 8b and a are R 1 and R 2 are each independently branched chain C6 to C 18 Alkyl or branched C6-C 18 fluoroalkyl].

[0099] In some embodiments, R 8a At least one occurrence of R is H. For example, in some embodiments, 8a is H at each occurrence. In some embodiments, R 8a or R 8b At least one occurrence of R is F. For example, in some embodiments, 8a is F for each occurrence. As another example, in some embodiments, R 8b is F for each occurrence.

[0100] In some embodiments, R 8a is C2 fluoroalkyl, R 8b is C alkyl and a is 1. In some embodiments, R 8a occurs once and is C2 fluoroalkyl; R 8b is H for each occurrence and a is 10. In some embodiments, R 8a is a C9 alkyl group, R 8b is F for two occurrences and a is 3.

[0101] In some embodiments, R 8a is C8 fluoroalkyl, R 8b is C fluoroalkyl and a is 1. In some embodiments, R 8a is a C8 fluoroalkyl group, R 8b is F with 5 occurrences and a is 9.

[0102] In some embodiments, R 1 , R 2 , or both are branched chain C6-C 18 For example, in some embodiments, R 1 , R 2 , or both are branched chain C 10 ~C 18 In some embodiments, R 1 Or R 2 or both have the structure: [ka] It has one of the following.

[0103] In some embodiments, R 1a and R 2a At least one of them is C6~C 24 Alkyl acetal or C6~C 24 For example, in some embodiments, R 1a and R 2a At least one of the following structures: [ka] has.

[0104] In some embodiments, L 1 and L 2 At least one of each is R 1b or R 2b For example, in some embodiments, R 1b Or R 2b or both have the structure: [ka] has.

[0105] In some embodiments, R 3 is OH. In some embodiments, R 3 is CN. In some embodiments, R 3 is C(=O)OR 4 ,OC(=O)R 4 or -NHC(=O)R 4 For example, in some embodiments, R 4 is methyl or ethyl.

[0106] In some embodiments, R 3 -C(=O)OR 4 , -C(=O)N(R 4 )R 5 , or -NR 5 C(=O)R 4 is [In the formula, R 4 and / or R 5 is hydroxy, aryl, OR 4a , O(C=O)R 4a , NH(C=O)R 4a where R is optionally replaced by 4a is C1-C6 alkyl optionally substituted with hydroxy. For example, in some embodiments, R 3 has the following structure: [ka] Have one of

[0107] In some embodiments, the compound has the following structure (IG) or (IH): [ka] Have one of [In the formula, R 11 and R 12are independent of each other, C1~C 12 is alkyl; or R 11 and R 12 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocyclic ring containing one nitrogen atom.

[0108] In some embodiments, the compound has the structure (IG): In other embodiments, the compound has the structure (IH).

[0109] In some embodiments, structure (IG) or (IH) has R 11 and R 12 In some embodiments, at least one of R in structure (IG) or (IH) is methyl. 11 and R 12 In some embodiments, structure (IG) or (IH) has R 11 and R 12 In some embodiments, at least one of R in structure (IG) or (IH) is ethyl. 11 and R 12 In some embodiments, structure (IG) or (IH) has R 11 and R 12 In some embodiments, structure (IG) or (IH) has R 11 and R 12 taken together with the nitrogen atom to form piperidine. In some embodiments, structure (IG) or (IH) has R 11 and R 12 together with the nitrogen atom to form an azepane.

[0110] In some embodiments, G 1 is a straight chain C1~C 12 In some embodiments, G is a fluoroalkylene. 2 is a straight chain C1~C 12 In some embodiments, R 1a Branched chain C6~C 24In some embodiments, R 2a Branched chain C6~C 24 It is a fluoroalkyl.

[0111] In some embodiments, the compound has at least two fluorine atoms. In some embodiments, the compound has at least three fluorine atoms. In some embodiments, the compound has at least one perfluoro substituent. In some embodiments, the compound is a perfluoro compound.

[0112] In various other embodiments, the compound has one of the structures presented in Table 1 below. Table 1. Representative compounds [Table 1] [Table 2]

[0113] It is understood that any embodiment of the compounds of structure (I) as described above, and any particular substituents and / or variables in the compounds of structure (I) as described above, can be independently combined with other embodiments of the compounds of structure (I) and / or substituents and / or variables to form embodiments of the invention not specifically described above. In addition, when a list of substituents and / or variables is recited in a particular embodiment and / or claim for any particular R group, G group, L group, or variable a, y, z, or n, it is understood that each individual substituent and / or variable may be deleted from the particular embodiment and / or claim, and the remaining list of substituents and / or variables is considered to be within the scope of the present disclosure.

[0114] It is understood that in this description, combinations of substituents and / or variables of the depicted formula are permissible only if such contributions result in stable compounds.

[0115] In some embodiments, compositions are provided that include a compound of structure (I). In some embodiments, compositions are provided that include lipid nanoparticles that include a compound of structure (I). The lipid nanoparticles optionally include an excipient selected from neutral lipids, steroids, and polymer-conjugated lipids.

[0116] In some embodiments, lipid nanoparticles are provided that comprise any one or more of the compounds of structure (I) and a therapeutic agent. For example, in some embodiments, the lipid nanoparticles comprise any one of the compounds of structure (I), a therapeutic agent, and one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids. Other pharma-ceutically acceptable excipients and / or carriers are also included in various embodiments of the lipid nanoparticles.

[0117] In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC. In various embodiments, the molar ratio of the compound to the neutral lipid ranges from about 2:1 to about 8:1.

[0118] In various embodiments, the lipid nanoparticle further comprises a steroid or a steroid analog. In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of the compound to cholesterol ranges from about 5:1 to 1:1 or 2:1 to 5:1.

[0119] In various embodiments, the polymer-conjugated lipid is a PEGylated lipid. For example, some embodiments include PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), and the like. ), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecaneoxy)propyl)carbamate, or 2,3-di(tetradecaneoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the compound to the PEGylated lipid is about 100:1 to about 20:1. In some embodiments, the molar ratio of the compound to the PEGylated lipid is about 100:1 to about 20:1, or about 100:1 to about 10:1.

[0120] In some embodiments, the lipid nanoparticle has the following structure (II): [ka] or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof. [In the formula, R 9 and R 10 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, where the alkyl chain is optionally interrupted by one or more ester bonds, and w has an average value of 30 to 60.

[0121] In some embodiments, R 9 and R 10are each independently a linear, saturated alkyl chain containing from 12 to 16 carbon atoms. In other embodiments, w has an average of from about 42 to 55, e.g., about 49.

[0122] In some embodiments of the above lipid nanoparticles, the therapeutic agent comprises a nucleic acid. For example, in some embodiments, the nucleic acid is selected from antisense RNA and messenger RNA.

[0123] In another alternative embodiment, the present disclosure is directed to a method for administering a therapeutic agent to a patient in need thereof, comprising preparing or providing any of the aforementioned compositions and administering the composition to the patient.

[0124] For purposes of administration, the compounds of the embodiments of the present disclosure (usually in the form of lipid nanoparticles combined with a therapeutic agent) may be administered as raw chemicals or may be formulated as pharmaceutical compositions. Pharmaceutical compositions in the embodiments of the present disclosure include a compound of structure (I) and one or more pharma- ceutically acceptable carriers, diluents, or excipients. In some embodiments, the compound of structure (I) is present in the composition in an amount that is effective to form lipid nanoparticles and deliver a therapeutic agent, for example, to treat a particular disease or condition of interest. Appropriate concentrations and dosages can be readily determined by one of skill in the art.

[0125] The administration of the compositions in the embodiments of the present disclosure may be via any of the accepted modes of administration of pharmaceuticals to perform the same utility. The pharmaceutical compositions in the embodiments of the present disclosure may be formulated into preparations in the form of solid, semi-solid, liquid or gas, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, intrabuccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions in the embodiments of the present disclosure are formulated so that the active ingredients contained therein are bioavailable when the compositions are administered to a patient. In some embodiments, the composition administered to the subject or patient is in the form of one or more dosage units, where for example, a tablet can be a single dosage unit, and a container of the compound in the embodiment of the present disclosure in the form of an aerosol can have multiple dosage units.The actual method of preparing such dosage forms is known or will be clear to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphia College of Pharmacy and Science, 2000).In some embodiments, the composition administered contains a therapeutically effective amount of the compound of the present disclosure or its pharma-ceutically acceptable salt for treating the disease or condition of interest, in any case according to the teachings of the present disclosure.

[0126] The pharmaceutical composition in the embodiment of the present disclosure may be in solid or liquid form. In one aspect, the carrier(s) are particulate, whereby the composition is, for example, in tablet or powder form. The carrier(s) may be liquid, whereby the composition is, for example, an oral syrup, an injectable solution, or an aerosol, which is useful, for example, for inhalation administration.

[0127] When intended for oral administration, the pharmaceutical compositions in certain embodiments are preferably in either solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms being included in forms that are considered herein to be either solid or liquid.

[0128] As a solid composition for oral administration, the pharmaceutical composition in some embodiments may be formulated into the form of powders, granules, compressed tablets, pills, capsules, chewing gums, wafers, etc. Such solid compositions will usually contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders, such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth or gelatin; excipients, such as starch, lactose or dextrin; disintegrants, such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants, such as magnesium stearate or Sterotex; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; flavorings, such as peppermint, methyl salicylate or orange flavoring, etc.; and coloring agents.

[0129] When the pharmaceutical composition in some embodiments is in the form of a capsule, for example, a gelatin capsule, the pharmaceutical composition may contain, in addition to the materials listed above, a liquid carrier, such as polyethylene glycol or oil.

[0130] In some embodiments, the pharmaceutical composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, the preferred composition contains, in addition to the compound of structure (I), one or more of a sweetener, a preservative, a dye / colorant, and a flavor enhancer. In a composition intended for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent may be included.

[0131] Liquid pharmaceutical compositions in the embodiments of the present disclosure, whether they are in a solution, suspension or other similar form, may contain one or more of the following adjuvants: sterile diluents, such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic mono- or diglycerides, polyethylene glycols, glycerin, propylene glycol or other solvents that can serve as a solvent or suspending medium; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and tonicity adjusters, such as sodium chloride or dextrose; agents that act as cryoprotectants, such as sucrose or trehalose. Parenteral preparations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Saline is the preferred adjuvant. Pharmaceutical compositions for injection are preferably sterile.

[0132] The pharmaceutical composition in the embodiment of the present disclosure may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base may, for example, comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Viscosifiers may be present in pharmaceutical compositions for topical administration. When intended for transdermal administration, the composition may comprise a transdermal patch or iontophoresis device.

[0133] The pharmaceutical composition in the embodiment of the present disclosure may contain various materials that modify the physical form of solid or liquid dosage units.For example, the composition may contain materials that form a coating shell around the active ingredient.The materials that form the coating shell are usually inert and can be selected from, for example, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredient can be placed in a gelatin capsule.

[0134] Pharmaceutical compositions in embodiments of the present disclosure, in solid or liquid form, may include agents that bind to the compounds of the present disclosure and thereby aid in the delivery of the LNP. Suitable agents that can act in this capacity include monoclonal or polyclonal antibodies, or proteins.

[0135] The pharmaceutical composition in the present disclosure may be comprised of a dosage unit that can be administered as an aerosol. The term aerosol is used to describe a variety of systems ranging from colloidal nature to systems comprised of pressurized packages. Delivery may be by liquefied or compressed gas or by a suitable pump system to dispense the active ingredient. The aerosol of the LNP in the present disclosure may be delivered in a single-phase, two-phase, or three-phase system to deliver the active ingredient(s). The delivery of the aerosol includes the necessary container, activator, valve, subcontainer, etc., which may together form a kit. Those skilled in the art can determine the preferred aerosol without undue experimentation.

[0136] The pharmaceutical composition in the embodiment of the present disclosure can be prepared by known methods in the pharmaceutical field.For example, pharmaceutical compositions intended for administration by injection can be prepared by combining the lipid nanoparticles of the present disclosure with sterile, distilled water or other carriers to form a solution.Surfactants can be added to promote the formation of a homogeneous solution or suspension.Surfactants are compounds that non-covalently interact with the compounds of the present disclosure to promote the dissolution or homogeneous suspension of the compounds in aqueous delivery systems.

[0137] The compositions, or pharma- ceutically acceptable salts thereof, in embodiments of the present disclosure are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular therapeutic agent employed, the metabolic stability and duration of action of the therapeutic agent, the age, weight, general health, sex, and dietary habits of the patient, the mode and time of administration, rate of excretion, drug combination, the severity of the particular disorder or condition, and the subject being treated.

[0138] The composition in the embodiments of the present disclosure may also be administered simultaneously with, before, or after administration of one or more other therapeutic agents. Such combination therapy includes administration of the composition in the embodiments of the present disclosure and one or more additional active agents in a single pharmaceutical dosage formulation, as well as administration of the composition in the embodiments of the present disclosure and the active agents in separate pharmaceutical dosage formulations. For example, the composition in the embodiments of the present disclosure and the other active agents can be administered to a patient together as a single oral dosage formulation, such as a tablet or capsule, or each agent can be administered as a separate oral dosage formulation. When separate dosage formulations are used, the compound in the embodiments of the present disclosure and one or more additional active agents can be administered at essentially the same time, i.e., simultaneously, or separately at different times, i.e., sequentially, and combination therapy is understood to include all of these regimens.

[0139] Methods of preparation for the above compounds and compositions are described herein below and / or are known in the art.

[0140] Those skilled in the art will appreciate that in the processes described herein, it may be necessary to protect the functional groups of intermediate compounds with suitable protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl, or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R'', where R'' is alkyl, aryl, or arylalkyl, p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl, or arylalkyl esters. Protecting groups can be added or removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3rd Edition., Wiley. As will be appreciated by those skilled in the art, the protecting group may also be a polymer resin, such as a Wang resin, a Rink resin, or a 2-chlorotrityl-chloride resin.

[0141] It is also understood by those skilled in the art that these protected derivatives of the compounds of the present disclosure may not possess pharmacological activity themselves, but may be administered to a mammal and then metabolized in the body to form the compounds of the present disclosure that are pharmacologically active. Thus, such derivatives may be described as "prodrugs." All prodrugs of the present disclosure are included within the scope of the present disclosure.

[0142] Additionally, compounds in embodiments of the present disclosure that exist in free base or acid form can be converted to their pharma- ceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid by methods known to those of skill in the art. Salts of compounds in embodiments of the present disclosure can be converted to their free base or free acid forms by standard techniques.

[0143] The following general reaction schemes 1 and 2 are useful for preparing compounds of the present invention, i.e., Structure (I), Structure (IC), or Structure (ID): [ka] 1 illustrates an exemplary method for synthesizing a compound having the formula: 1a , R 2a , R 3 , L 1 , L 2 , G 1 , G 2 , and G 3 are as defined herein]. It is understood that those skilled in the art may synthesize these compounds in a similar manner or by combining other methods known to those skilled in the art. It is also understood that those skilled in the art may synthesize other structures (I), (IC), or (ID) not specifically illustrated below in a similar manner to those described below by using appropriate starting components and modifying the synthetic parameters as necessary. In general, the starting components may be obtained from commercial sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, may be synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition (Wiley, December 2000)), or may be prepared as described in this disclosure.

[0144] [ka] General reaction scheme 1 shows the reaction of L 1 and L 2 are -O(C=O)R 1a and -O(C=O)R 2a An exemplary method for preparing a compound of structure (IC) is provided. 1 , G 3 , R 1a , R 2a , and R 3 is as defined herein, and G 1’ is G 1 A-1 refers to the analogue with one less carbon. Compounds of structure A-1 are either purchased or prepared by methods known in the art. Reaction of A-1 with diol A-2 under suitable condensation conditions (e.g. DCC) gives ester / alcohol A-3, which can then be oxidized (e.g. PCC) to aldehyde A-4. Reaction of A-4 with amine A-5 under reductive amination conditions gives compounds of structure (IC).

[0145] [ka] General reaction scheme 2 shows the reaction of L 1 and L 2 are -(C=O)OR 1a and -(C=O)OR 2a An exemplary method for preparing a compound of structure (ID) is provided. 1 , G 3 , R 1a , R 2a , and R 3are as defined herein. Compounds of structure A-6 are either commercially available or prepared by methods known in the art. A-6 can be other leaving groups such as iodide, chloride, tosylate, triflate, etc. Reaction of A-6 with alcohol A-7 under suitable esterification conditions under acidic conditions gives ester A-8. Reaction of A-8 with amine A-5 under reductive amination conditions gives compounds of structure (ID).

[0146] It should be noted that various alternative strategies for preparing compounds of structure (IC) or (ID) are available to those skilled in the art. For example, L 1 and L 2 Other compounds of structure (IC) or (ID) where G is not an ester may be prepared following similar methods using appropriate starting materials. Additionally, General Reaction Schemes 1 and 2 show the general reaction schemes of G 1 and G 2 Although the present invention depicts the preparation of compounds of structures (IC) and (ID), in which G is the same, this is not a required aspect of the invention, and the above reaction scheme can be modified to provide 1 and G 2 In addition, it is possible to obtain compounds with different R 1a and R 2a While the present invention depicts the preparation of compounds of structures (IC) and (ID), in which R 1a and R 2a It is possible to obtain compounds that differ in structure. The use of necessary protecting groups and other modifications to the above general reaction schemes will be readily apparent to one of ordinary skill in the art. The following examples are offered for purposes of illustration and not limitation. EXAMPLES

[0147] In vivo evaluation of luciferase mRNA using lipid nanoparticle compositions Lipid nanoparticles were prepared and tested according to the general procedures described in PCT Publication Nos. WO2015 / 199952 and WO2017 / 004143, the entire disclosures of which are incorporated herein by reference. Briefly, cationic lipid, DSPC, cholesterol, and PEG lipids were solubilized in ethanol at a molar ratio of about 50:10:38.5:1.5 or at a molar ratio of about 47.5:10:40.7:1.8. Lipid nanoparticles (LNPs) were prepared at a total lipid to mRNA weight ratio of about 10:1 to 40:1. The mRNA was diluted to 0.2 mg / mL in 10-50 mM citrate or acetate buffer at pH 4. A syringe pump was used to mix the ethanolic lipid solution with the aqueous mRNA solution at a ratio of about 1:5 to 1:3 (vol / vol) at a total flow rate of greater than 15 mL / min. The ethanol was then removed and the external buffer was replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 μm pore sterile filter. The lipid nanoparticles had a diameter of about 55-95 nm, and in some cases about 70-90 nm, as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK).

[0148] Studies were performed in 6-8 week old female C57BL / 6 mice (Charles River) or 8-10 week old CD-1 (Harlan) mice (Charles River) in accordance with guidelines established by the institutional animal care committee (ACC) and the Canadian Council on Animal Care (CCAC). Various doses of mRNA-lipid nanoparticles were administered systemically via tail vein injection, and animals were euthanized at specific time points (e.g., 4 hours) after administration. Livers and spleens were collected in pre-weighed tubes, weighed, immediately flash frozen in liquid nitrogen, and stored at -80°C until processed for analysis.

[0149] Approximately 50 mg of liver was dissected for analysis and placed into 2 mL FastPrep tubes (MP Biomedicals, Solon, OH). ¼ inch ceramic balls (MP Biomedicals) were added to each tube, and 500 μL of Glo Lysis Buffer-GLB (Promega, Madison WI) equilibrated to room temperature was added to the liver tissue. The liver tissue was homogenized using a FastPrep24 instrument (MP Biomedicals) at 2 × 6.0 m / sec for 15 seconds. The homogenate was incubated at room temperature for 5 minutes before being diluted 1:4 in GLB and evaluated using the SteadyGlo Luciferase assay system (Promega). Specifically, 50 μL of the diluted tissue homogenate was reacted with 50 μL of SteadyGlo substrate, shaken for 10 seconds, followed by incubation for 5 minutes, and then assayed using a CentroXS. 3 Quantification was performed using an LB 960 luminometer (Berthold Technologies, Germany). The amount of assayed protein was determined using a BCA protein assay kit (Pierce, Rockford IL). Relative luminescence units (RLU) were then normalized to total protein assayed (μg). To convert RLU to luciferase (ng), a standard curve was generated using QuantiLum Recombinant Luciferase (Promega).

[0150] Trilink Biotechnologies' FLuc mRNA (L-6107 or L-7202) expresses the luciferase protein originally isolated from the firefly, photinus pyralis. FLuc is commonly used in mammalian cell culture to measure both gene expression and cell viability. It produces bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA is fully substituted for uridine and / or cytidine nucleosides. EXAMPLES

[0151] In vivo evaluation of immunoglobulin G (IgG) mRNA using lipid nanoparticle compositions The lipids of structure (I), DSPC, cholesterol and PEGylated lipids are solubilized in ethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.7:1.8. Lipid nanoparticles (LNPs) are prepared at a total lipid to mRNA weight ratio of about 10:1 to 40:1. Briefly, the mRNA is diluted to 0.2 mg / mL in 10-50 mM citrate buffer (pH 4) or 10-25 mM acetate buffer (pH 4). Using a syringe pump, the ethanolic lipid solution and the aqueous mRNA solution are mixed at a ratio of about 1:5 to 1:3 (vol / vol) at a total flow rate of more than 15 mL / min. The ethanol is then removed and the external buffer is replaced by PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 μm pore sterile filter.

[0152] Studies are performed in 6-8 week old CD-1 / ICR mice (Envigo) following guidelines established by the institutional animal care committee (ACC) and the Canadian Council on Animal Care (CCAC). Different doses of mRNA-lipid nanoparticles are administered systemically via tail vein injection and animals are euthanized at specific time points (e.g., 24 hours) after administration. Whole blood is collected and serum is subsequently separated by centrifugation of the whole blood tubes at 2000×g for 10 minutes at 4°C and stored at -80°C until processed for analysis.

[0153] For Immunoglobulin G (IgG) ELISA (Life Diagnostics Human IgG ELISA Kit), dilute the serum samples 100-15000 times with 1x diluent. Dispense 100 μL of diluted serum into an anti-human IgG coated 96-well plate and in parallel incubate the human IgG standard at 25°C for 45 minutes at 150 rpm on a plate shaker. Wash wells 5 times with 1x wash solution using a plate washer (400 μL / well). Add 100 μL of HRP conjugate to each well and incubate on a plate shaker under the same conditions as above. Wash wells again 5 times with 1x wash solution using a plate washer (400 μL / well). Add 100 μL of TMB reagent to each well and incubate on a plate shaker under the same conditions as above. Terminate the reaction by adding 100 μL of stop solution to each well. The absorbance at 450 nm (A450) is read using a microplate reader. The A450 value of the standard assay is plotted against the human IgG concentration to determine the amount of human IgG in the mouse serum. EXAMPLES

[0154] pKa Determination of Formulated Lipids As described elsewhere, the pKa of the formulated cationic lipid correlates with the efficacy of the LNP for nucleic acid delivery (see Jayaraman et al., Angewandte Chemie, International Edition (2012), vol. 51(34), pp. 8529-8533; Semple et al., Nature Biotechnology vol. 28, pp. 172-176 (2010)). The preferred range of pKa is about 5 to about 7. The pKa of each cationic lipid was determined in lipid nanoparticles using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles containing cationic lipid / DSPC / cholesterol / PEG-lipid (47.5 / 10 / 40.7 / 1.8 mol%) in PBS at a total lipid concentration of 0.4 mM were prepared using the in-line process described in Example 1. TNS was prepared as a stock solution of 100 μM in distilled water. Vesicles were diluted with 24 μM lipid / 2 mL buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl (pH range 2.5-11). Aliquots of TNS solution were added to a final concentration of 1 μM, vortex mixed, and fluorescence intensity was measured at room temperature using excitation and emission wavelengths of 321 nm and 445 nm on an SLM Aminco Series 2 Luminescence Spectrophotometer. A sigmoidal best-fit analysis was applied to the fluorescence data to determine the pKa as the pH at which half-maximal fluorescence intensity was observed. EXAMPLES

[0155] Determining the efficacy of lipid nanoparticle formulations containing different cationic lipids using a rodent model expressing luciferase / IgG mRNA in vivo Representative compounds of the disclosure shown in Table 2 were formulated using the following molar ratios: 50% cationic lipid / 10% distearoylphosphatidylcholine (DSPC) / 38.5% cholesterol / 1.5% PEG lipid 2-[2-(ω-methoxy(polyethylene glycol) 2000)ethoxy]-N,N-ditetradecylacetamide) or 47.5% cationic lipid / 10% DSPC / 40.7% cholesterol / 1.8% PEG lipid. Relative activity was determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection as described in Example 1, or by measuring the amount of human IgG in mouse serum as described in Example 2. Activity was compared when the mRNA was dosed at 1.0 mg / kg, 0.5 mg / kg, or 0.3 mg / kg, and activity was expressed as luciferase (ng) / liver (g) measured 4 hours after administration as described in Example 1, or IgG (μg) / serum (mL) measured 24 hours after administration as described in Example 2. Compound numbers in Table 2 refer to compound numbers in Table 1. Table 2. Cationic lipids and their associated activities [Table 3] The I-3 analog is a non-fluorinated analog of compound I-3 and has the following structure: [ka] has. EXAMPLES

[0156] Bis(1,1,1,2,2-pentafluorododecan-3-yl) 8,8'-((2-hydroxyethyl)azanediyl) dioctanoate (compound I-1) [ka] Synthesis of 1,1,1,2,2-pentafluorododecan-3-yl 8-bromooctanoate (Intermediate A) Dicyclohexylcarbodiimide (DCC) (1.1 g, 5.33 mmol) was added to a round-bottom flask filled with anhydrous DCM solution of 1,1,1,2,2-pentafluorododecan-3-ol (0.70 g, 2.53 mmol), 8-bromooctanoic acid (1.13 g, 5.06 mmol), and 4-dimethylaminopyridine (DMAP) (60 mg). The precipitate was removed by filtration. The filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography (eluted with ethyl acetate / hexane mixture (concentration gradient: 0-3%)). This gave 1,1,1,2,2-pentafluorododecan-3-yl 8-bromooctanoate as a colorless oil (1.00 g, 2.08 mmol, 82%).

[0157] Synthesis of I-1 To a solution of 2-aminoethanol (33 mg, 0.55 mmol) in anhydrous THF (15 mL) was added 1,1,1,2,2-pentafluorododecan-3-yl 8-bromooctanoate (0.50 g, 1.04 mmol), potassium carbonate (0.14 g, 1.04 mmol), cesium carbonate (53 mg, 0.16 mmol) and sodium iodide (10 mg). The mixture was heated to reflux under nitrogen for 7 days. The solvent was evaporated under reduced pressure and the residue was dissolved in hexane / EtOAc (9:1) and washed with water and saturated brine. The organic layer was separated, dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure to give a colorless oil. The crude product was purified several times by silica gel column chromatography (0-5% MeOH / DCM gradient) to give I-1 as a colorless oil (50 mg, 0.06 mmol, 11%). 1 HNMR (400 MHz, CDCl3) δ: 5.51-5.37 (m, 2H), 3.53 (t, 5.4 Hz, 2H), 2.58 (t, 5.4 Hz, 2H), 2.45 (t, 7.4 Hz, 4H), 2.37 (t, 7.4 Hz, 4H), 1.81-1.54 (m, 8H), 1.48-1.38 (m, 4H) 1.37-1.19 (m, 40H), 0.88 (t, 7.0 Hz, 6H). ESI-MS: C 42 H73 F 10 NO5[M+H] + MW calculated 862.5; measured 862.7. EXAMPLES

[0158] Bis(1,1,1,2,2-pentafluorododecan-3-yl) 6,6'-((3-hydroxypropyl)azanediyl) dihexanoate (compound I-2) Compound I-2 was prepared according to the general procedure described in Example 5 to yield 0.16 g of a colorless oil (0.19 mmol, 18%). 1 HNMR (400 MHz, CDCl3)δ: 5.53-5.38 (m, 2H), 3.80 (t, 5.2 Hz, 2H), 2.64 (t, 5.2 Hz, 2H), 2.45-2.35 (m, 8H), 1.83-1.72 (m, 4H), 1.72-1.62 (m, 6H), 1.55-1.45 (m, 4H), 1.39-1.20 (m, 32H), 0.89 (t, 7.2 Hz, 6H). ESI-MS: C 39 H 67 F 10 NO5[M+H] + MW calculated 820.5; measured 820.6. EXAMPLES

[0159] Bis(1,1,1,2,2-pentafluorododecan-3-yl) 6,6'-((4-hydroxybutyl)azanediyl) dihexanoate (compound I-3) Compound I-3 was prepared according to the general procedure described in Example 5 to yield 0.18 g of a colorless oil (0.21 mmol, 16%). 1HNMR (400 MHz, CDCl3) δ: 5.50-5.37 (m, 2H), 3.57-3.50 (m, 2H), 2.48-2.33 (m, 10H), 1.84-1.56 (m, 12H), 1.55-1.44 (m, 4H), 1.39-1.18 (m, 32H), 0.88 (t, 7.0 Hz, 6H). ESI-MS: C 40 H 69 F 10 NO5[M+H] + MW calculated 834.5; measured 834.7. EXAMPLES

[0160] Bis(1,1,1,2,2-pentafluorododecan-3-yl) 6,6'-((5-hydroxypentyl)azanediyl)dihexanoate (Compound I-4) Compound I-4 was prepared according to the general procedure described in Example 5 to yield 85 mg of an oil (0.10 mmol, 12%). 1 HNMR (400 MHz, CDCl3) δ: 5.50-5.37 (m, 2H), 3.65 (t, 6.4 Hz, 2H), 2.61-2.42 (m, 6H), 2.38 (t, 7.1 Hz, 4H), 1.84-1.45 (m, 14H), 1.43-1.19 (m, 36H), 0.88 (t, 7.0 Hz, 6H). ESI-MS: C 41 H 71 F 10 NO5[M+H] + MW calculated 848.5; measured 848.7. EXAMPLES

[0161] Bis(1,1,1,2,2,3,3,4,4,5,5,6,6,12,12,13,13,14,14,15,15,16,16,17,17,17-hexacosafluoroheptadecan-9-yl) 6,6'-((4-hydroxybutyl)azanediyl) dihexanoate (compound I-5) [ka] Synthesis of 1,1,1,2,2,3,3,4,4,5,5,6,6,12,12,13,13,14,14,15,15,16,16,17,17,17-Hexacosafluoroheptadecan-9-ol (Intermediate B) 1H,1H,2H,2H-Perfluorooctyl iodine (5 g, 10.55 mmol) was added slowly to an ice-cold solution of isopropylmagnesium chloride (2.0 M / THF, 4.5 mL) and anhydrous THF (10 mL), maintaining the internal temperature below 15 °C. After 20 min, ethyl formate (0.35 mL, 4.36 mmol) was added over 5 min. The ice-water bath was removed and the reaction mixture was stirred for 30 min. The reaction flask was cooled again on an ice-water bath and chilled HCl solution (1N, 20 mL) was added slowly. Extraction with Et2O was performed, the organic layer was washed with aqueous Na2SO4, dried over MgSO4, filtered and evaporated under reduced pressure to give a white solid. Recrystallization from methylene chloride gave pure 1,1,1,2,2,3,3,4,4,5,5,6,6,12,12,13,13,14,14,15,15,16,16,17,17,17-hexacosafluoroheptadecan-9-ol (3.5 g, 4.83 mmol, 91%).

[0162] 1,1,1,2,2,3,3,4,4,5,5,6,6,12,12,13,13,14,14,15,15,16,16,17,17,17-Hexacosafluoroheptadecan-9-yl 6-bromohexanoate (Intermediate C) To a round bottom flask charged with a solution of intermediate B (1.09 g, 1.50 mmol), 6-bromooctanoic acid (0.59 g, 3.00 mmol) and 4-dimethylaminopyridine (DMAP) (0.28 g) in anhydrous THF was added dicyclohexylcarbodiimide (DCC) (0.65 g, 3.15 mmol). The mixture was stirred at room temperature overnight. The solid was then filtered and washed with EtO. The filtrate was concentrated. The residue was filtered through a silica pad, washed with EtO and concentrated to give a white solid (1.20 g, 1.33 mmol) which was used in the next step without further purification.

[0163] Synthesis of I-5 To a solution of 4-aminobutan-1-ol (74 mg, 0.83 mmol) in anhydrous THF was added intermediate C (1.20 g, 1.33 mmol), potassium carbonate (0.22 g, 1.57 mmol), cesium carbonate (81 mg, 0.25 mmol) and sodium iodide (10 mg). The mixture was heated to reflux under nitrogen for 7 days. The solvent was evaporated under reduced pressure and the residue was dissolved in DCM and washed with saturated NaHCO3 solution, water and saturated brine. The organic layer was separated, dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The crude product was purified several times by silica gel column chromatography (0-5% MeOH / DCM gradient) to give I-5 as a colorless oil (40 mg, 0.02 mmol, 3.0%). 1 HNMR (400 MHz, CDCl3) δ: 5.00 (quint, 6.1 Hz, 2H), 3.56-3.50 (m, 2H), 2.44-2.37 (m, 6H), 2.34 (t, 7.4 Hz, 4H), 2.20-2.02 (m, 8H), 1.97-1.81 (m, 8H), 1.70-1.43 (m, 12H), 1.35-1.23 (m, 4H). EXAMPLES

[0164] 8,8'-((2,2-difluoro-3-hydroxypropyl)azanediyl)bis(N,N-didecyl octanamide) (Compound I-6) [ka] 8-Bromo-N,N-didecyl octanamide (Intermediate D) To a solution of 8-bromooctanoic acid (1 eq. 10.78 mmol, 2.41 g) in DCM (20 mL) and DMF (d 0.944; 0.1 mL) was added oxalyl chloride (2.5 eq. 27 mmol, 3.42 g, 2.35 mL) at room temperature under argon. The resulting mixture was stirred at room temperature overnight. The mixture was then concentrated under reduced pressure. The residue was dissolved in 15 mL of DCM and slowly added to a solution of didecylamine (1.1 eq. 3.53 g, 11.86 mmol), triethylamine (53.9 mmol, 7.5 mL) and DMAP (10 mg) in DCM (20 mL) at room temperature. Upon completion of the addition, the mixture was stirred at room temperature overnight and then concentrated. The residue was dissolved in hexane (100 mL) and loaded onto a silica gel column under reduced pressure. The column was then washed with a mixture of hexane and ethyl acetate (100:0 to 90:10) under reduced pressure to give the desired product as a yellow oil (4.81 g, 9.6 mmol, 89%).

[0165] Synthesis of I-6 A mixture of intermediate D (1.0 mmol, 500 mg), 3-amino-2,2-difluoropropan-1-ol (0.62 mmol, 69 mg), and DIEA (3.0 mmol, 0.43 mL) in ACN (6 mL) was heated at 72° C. for 48 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 60% EtOAc / hexanes with 1% EtN) to give compound I-6 (55 mg, 12%). 1 H NMR (600 MHz, CDCl3) δ 4.87 (s, 1H), 3.88 (t, J = 12.1 Hz, 2H), 3.33 - 3.27 (m, 4H), 3.24 - 3.18 (m, 4H), 2.96 (t, J = 12.7 Hz, 2H), 2.56 - 2.51 (m, 4H), 2.32 - 2.26 (m, 4H), 1.68 - 1.61 (m, 6H), 1.59 - 1.42 (m, 12H), 1.39 - 1.23 (m, 70H), 0.93 - 0.87 (m, 12H). ESI-MS: C 59 H 117F2N3O3[M+H] + MW calculated 954.9; measured 955.1. EXAMPLES

[0166] 8,8'-((2-fluoro-3-hydroxypropyl)azanediyl)bis(N,N-didecyl octanamide) (Compound I-7) [ka] Synthesis of I-7 A mixture of intermediate D (1.0 mmol, 500 mg), 3-amino-2-fluoropropan-1-ol hydrochloride (0.62 mmol, 80 mg), and DIEA (3.1 mmol, 0.54 mL) in ACN (6 mL) was heated at 72 °C for 24 h, then at 55 °C for 72 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 60% EtOAc / hexanes with 1% EtN) to give compound I-7 (45 mg, 10%). 1 H NMR (400 MHz, CDCl3) δ 4.60 (dt, J = 46.8, 4.8 Hz, 1H), 3.92 - 3.80 (m, 2H), 3.35 - 3.24 (m, 4H), 3.24 - 3.12 (m, 4H), 2.85 - 2.72 (m, ESI-MS: C59H 118 FN3O3[M+H] + MW calculated 936.9; measured 937.0. EXAMPLES

[0167] 8,8'-((3,3,3-trifluoro-2-(hydroxymethyl)propyl)azanediyl)bis(N,N-didecyl octanamide) (Compound I-8) [ka] Synthesis of I-8 A mixture of intermediate D (1.0 mmol, 500 mg), 2-(aminomethyl)-3,3,3-trifluoropropan-1-ol (0.62 mmol, 111 mg), and DIEA (2.4 mmol, 0.43 mL) in ACN (6 mL) was heated at 72 °C for 24 h, then at 55 °C for 72 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 60% EtOAc / hexanes with 1% EtN) to give compound I-8 (100 mg, 20%). 1 H NMR (400 MHz, CDCl3) δ 5.88 (s, 1H), 4.01 - 3.92 (m, 1H), 3.89 - 3.78 (m, 1H), 3.33 - 3.24 (m, 4H), 3.23 - 3.15 (m, 4H), 2.91 - 2.73 ESI-MS: C 60 H 118 F3N3O3[M+H] + MW calculated 986.9; measured 987.0. EXAMPLES

[0168] 8,8'-((5-hydroxypentyl)azanediyl)bis(N,N-didecyl-2-fluorooctanamide) (Compound I-9) [ka] Synthesis of diethyl 2-(6-bromohexyl)-2-fluoromalonate A mixture of diethyl 2-fluoromalonate (56.1 mmol, 10.0 g), 1,6-dibromohexane (168 mmol, 26 mL), and sodium methoxide (61.7 mmol, 3.3 g) in EtOH (110 mL) was stirred at room temperature for 24 h. The reaction mixture was concentrated and the crude product was partitioned between DCM and water. The organic layer was separated, dried over Na2SO4, filtered and concentrated. Purification by automated flash chromatography (0% to 25% EtOAc / hexane) gave diethyl 2-(6-bromohexyl)-2-fluoromalonate (11.7 g, 61%).

[0169] Synthesis of 2-(6-bromohexyl)-2-fluoromalonic acid A mixture of diethyl 2-(6-bromohexyl)-2-fluoromalonate (5.9 mmol, 2.0 g) and KOH (11.8 mmol, 660 mg) in MeOH (20 mL), THF (2 mL), and water (4 mL) was stirred at room temperature for 3 h. The reaction mixture was concentrated and the crude product was diluted with 0.1 M NaOH (20 mL). The aqueous layer was washed with DCM (3×10 mL), acidified with 1 M HCl, and then extracted with EtOAc (2×20 mL). The combined EtOAc layers were dried over Na2SO4, filtered, and concentrated to give 2-(6-bromohexyl)-2-fluoromalonic acid (1.57 g, 94%), which was used in the next step without further purification.

[0170] Synthesis of 8-bromo-2-fluorooctanoic acid A mixture of 2-(6-bromohexyl)-2-fluoromalonic acid (4.1 mmol, 1.2 g) and DMAP (catalytic amount) in DMF (3 mL) was heated at 180° C. for 12 min. The reaction mixture was partitioned between EtOAc and 1 M HCl. The organic layer was separated, dried over Na2SO4, filtered and concentrated to give 8-bromo-2-fluorooctanoic acid (960 mg, 98%), which was used in the next step without further purification.

[0171] Synthesis of 8-bromo-2-fluorooctanoyl chloride A mixture of 8-bromo-2-fluorooctanoic acid (4.0 mmol, 960 mg), oxalyl chloride (12 mmol, 1.0 mL), and DMF (catalytic amount) in DCM (10 mL) was stirred at room temperature for 20 min. The reaction mixture was concentrated to give 8-bromo-2-fluorooctanoyl chloride, which was used in the next step without further purification.

[0172] 8-Bromo-N,N-didecyl-2-fluorooctanamide To a mixture of didecylamine (4.0 mmol, 1.2 g), triethylamine (24 mmol, 3.4 mL), and DMAP (catalytic amount) in DCM (10 mL) was added a solution of the crude product 8-bromo-2-fluorooctanoyl chloride (4.0 mmol) in DCM (5 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction was concentrated and purified by automated flash chromatography (5% to 25% EtOAc / Hexanes) to give 8-bromo-N,N-didecyl-2-fluorooctanamide (1.2 g, 58% over two steps).

[0173] Synthesis of I-9 A mixture of 8-bromo-N,N-didecyl-2-fluorooctanamide (0.56 mmol, 290 mg), 5-aminopentanol (0.34 mmol, 35 mg), DIEA (1.0 mmol, 0.18 mL), potassium iodide (0.56 mmol, 93 mg) in ACN (4 mL) was heated at 75° C. for 19 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 100% EtOAc / hexanes) to give compound I-9 (158 mg, 57%). 1H NMR (400 MHz, CDCl3) δ 5.03 (ddd, J = 49.4, 8.5, 4.3 Hz, 2H), 3.64 (t, J = 6.5 Hz, 2H), 3.37 - 3.25 (m, 6H), 3.25 - 3.12 (m, 2H), 2.43 - 2.33 (m, 6H), 1.97 - 1.68 (m, 6H), 1.64 - 1.34 (m, 26H), 1.33 - 1.22 (m, 62H), 0.93 - 0.83 (m, 12H). ESI-MS: C 61 H 121 F2N3O3[M+H] + MW calculated 982.9; measured 983.0. EXAMPLES

[0174] 8,8'-(Methylazanediyl)bis(N,N-didecyl-2-fluorooctanamide) (Compound I-10) [ka] Synthesis of I-10 A mixture of 8-bromo-N,N-didecyl-2-fluorooctanamide (prepared according to the general procedure described in Example 13, 0.58 mmol, 300 mg), 8 M methylamine / EtOH (0.36 mmol, 0.045 mL), and DIEA (1.1 mmol, 0.19 mL) in ACN (4 mL) was heated at 75° C. for 48 h. The reaction mixture was concentrated and the crude product was purified by automated flash chromatography (5% to 65% EtOAc / Hexanes) to give compound I-10 (160 mg, 61%). 1 H NMR (400 MHz, CDCl3) δ 5.14 - 4.93 (m, 2H), 3.40 - 3.24 (m, 6H), 3.24 - 3.10 (m, 2H), 2.33 - 2.25 (m, 4H), 2.19 (s, 3H), 1.99 - 1.71 (m, 4H), 1.62 - 1.17 (m, 80H), 0.92 - 0.84 (m, 12H). ESI-MS: C 57 H 113F2N3O2[M+H] + MW calculated 910.9; measured 911.0.

[0175] Various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent documents referenced in this specification and application data sheets, including U.S. Provisional Patent Application No. 63 / 290,396, filed December 16, 2021, are incorporated herein by reference in their entirety. Aspects of the embodiments may be modified as necessary to utilize concepts from various patents, applications, and documents to provide further embodiments. These and other changes may be made to the embodiments in light of the description detailed above. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. The claims are therefore not limited by this disclosure.

Claims

1. The following structure (I): 【Chemistry 1】 (I) or a pharmaceutically acceptable salt or stereoisomer thereof [In the formula, L １ is -O(C=O)R １ａ , -(C=O)OR １ａ , -C(=O)R １ａ , -OR １ａ , -S(O) ｘ R １ａ , -S-SR １ａ , -C(=O)SR １ａ , -SC(=O)R １ａ , -NR ａ C(=O)R １ａ , —C(═O)NR ａ R １ａ , -NR ａ C(=O)NR ａ R １ａ , -OC(=O)NR ａ R １ａ , -NR ａ C(=O)OR １ａ or R １ｂ and L ２ is -O(C=O)R ２ａ , -(C=O)OR ２ａ , -C(=O)R ２ａ , -OR ２ａ , -S(O) ｘ R ２ａ , -S-SR ２ａ , -C(=O)SR ２ａ , -SC(=O)R ２ａ , -NR ａ C(=O)R ２ａ , —C(═O)NR ａ R ２ａ , -NR ａ C(=O)NR ａ R ２ａ , -OC(=O)NR ａ R ２ａ , -NR ａ C(=O)OR ２ａ , or R ２ｂ and G １ and G ２ are each independently a straight-chain or branched-chain C １ ~C １２ Alkylene or straight or branched chain C １ ~C １２ is a fluoroalkylene; G ３ is a straight or branched chain C optionally substituted with C 1 -C 24 alkyl １ ~C １２ Alkylene or straight or branched chain C １ ~C １２ is a fluoroalkylene; R ａ are each independently H or C １ ~C １２ is alkyl; R １ａ and R ２ａ are each independently a branched chain C ６ ~C ２４ Alkyl, branched chain C ６ ~C ２４ Fluoroalkyl, C ６ ~C ２４ Alkyl acetal, or C ６ ~C ２４ fluoroalkyl acetal; R １ｂ and R ２ｂ are each independently —CH(OR)(OR), where each R is independently a straight or branched chain C ６ ~C １８ Alkyl, straight or branched chain C ６ ~C １８ Alkenyl, straight or branched chain C ６ ~C １８ Fluoroalkyl, or linear or branched C ６ ~C １８ is fluoroalkenyl; R ３ is H, -OR ５ , -CN, -C(=O)OR ４ , —OC(═O)R ４ , -N(R ５ ) R ４ , -C(=O)N(R ４ ) R ５ , or -NR ５ C(=O)R ４ and R ４ is H, C １ ~C １２ alkyl, or aryl; R ５ is H or C １ ~C ６ alkyl; or R ４ and R ５ together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycle, wherein R 4 and R 5 are optionally substituted with one or more substituents selected from OH, oxo, aryl, amide, ester, and alkoxy optionally substituted with OH; and Here, G １ and G ２ At least one of the following is a linear or branched chain C １ ~C １２ fluoroalkylene; G ３ is a straight or branched chain C １ ~C １２ fluoroalkylene; R １ａ and R ２ａ At least one of the branched chain C ６ ~C ２４ Fluoroalkyl, and C ６ ~C ２４ fluoroalkyl acetals; and / or R １ｂ and R ２ｂ At least one of the following is present: ６ ~C １８ Fluoroalkyl and linear or branched C ６ ~C １８ fluoroalkenyl].

2. Structure (IA): 【Chemistry 2】 (IA) [In the formula, R ６ is, at each occurrence, independently H, F, OH, or C １ ~C ２４ is alkyl; and and n is an integer from 1 to 15. or Structure (IB): 【Transformation 3】 (IB) [In the formula, y and z are each independently an integer from 1 to 12; and R 7 at each occurrence is independently H or F.

2. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, having the formula:

3. The following structure (IE) or (IF): 【Chemistry 4】 3. The compound of claim 2, having one of:

4. a) n is an integer from 2 to 12, preferably n is 2, 3, 4, or 5; or b) y and z are each independently an integer from 2 to 10, preferably y and z are each independently an integer from 4 to 9; or c) R 6 is H; 3. The compound of claim 2, or a pharmaceutically acceptable salt or stereoisomer thereof.

5. L １ Ga-NR ａ C(=O)R １ａ or —C(═O)NR ａ R １ａ and L ２ Ga-NR ａ C(=O)R ２ａ or —C(═O)NR ａ R ２a Preferably, R ａ are C １ ~C １２ The compound of claim 1 , wherein the aryl group is alkyl.

6. R １ａ and R ２ａ each independently represents the following structure: 【Transformation 5】 [In the formula, R ８ａ and R ８ｂ are independently H, F, and C for each occurrence. ２ ~C １６ Alkyl, or C ２ ~C １６ is fluoroalkyl; a is an integer from 1 to 16; and R ８ａ , R ８ｂ and a are R １ and R ２ are each independently a branched chain C ６ ~C １８ Alkyl or branched chain C ６ ~C １８ selected to include fluoroalkyl] and preferably having a) R ８ａ at least one occurrence of is H; or b) R ８ａ is H for each occurrence; or c) R ８ａ or R ８ｂ at least one occurrence of is F; or d) R ８ａ is F for each occurrence, 10. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.

7. R １ａ , R ２ａ , or both are branched chain C ６ ~C １８ fluoroalkyl, preferably R 1a , R 2a , or both, are branched C 10 -C 18 fluoroalkyl; or R 1a or R 2a , or both, have the following structure: 【Transformation 6】 having one of 10. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.

8. R ３ is OH, CN or the following structure: 【Transformation 7】 10. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, having one of:

9. The following structure (IG) or (IH): 【Transformation 8】 [In the formula, R １１ and R １２ are each independently １ ~C １２ alkyl; or R １１ and R １２ together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocyclic ring containing one nitrogen atom.

10. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, having one of:

10. a) R １１ and R １２ at least one of which is methyl; b) R １１ and R １２ at least one of which is ethyl; c) R １１ and R １２ together with the nitrogen atom to form pyrrolidine; d) R １１ and R １２ together with the nitrogen atom to form piperidine; or e) R １１ and R １２ together with the nitrogen atom to form an azepane 10. The compound of claim 9, or a pharmaceutically acceptable salt or stereoisomer thereof.

11. a) G １ is a linear C １ ~C １２ is a fluoroalkylene; or b) G ２ is a linear C １ ~C １２ is a fluoroalkylene; or c) R １ａ is branched chain C ６ ~C ２４ is fluoroalkyl; or d) R ２ａ is branched chain C ６ ~C ２４ is a fluoroalkyl; 10. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.

12. 2. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, having at least two fluorine atoms, having at least three fluorine atoms, or having at least one perfluoro substituent, preferably a perfluoro compound.

13. A compound having one of the following structures, or a pharmaceutically acceptable salt or stereoisomer thereof: Table 1 Table 2

14. 14. A lipid nanoparticle or composition comprising the compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt or stereoisomer thereof, and a therapeutic agent, wherein preferably the therapeutic agent comprises a nucleic acid, and more preferably the nucleic acid is selected from antisense RNA and messenger RNA.

15. A lipid nanoparticle or composition as described in claim 14 for use in a method of treatment.

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